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QP601  .C66  Enzymes,  by  Otto  Coh 


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ENZYMES 


BY 

OTTO  COHNHEIM 

a.  o.  Professor  of  Physiology,  Heidelberg 


SIX  LECTURES 
Delivered  under  the  Herter  Lectureship  Foundation 

AT    THE 

University  and  Bellevue  Hospital  Medical  College 


FIRST  EDITION 

Firs  r  Thousand 


NEW  YORK: 
JOHN  WILEY  &  SONS 

London:   CHAPMAN  &  HALL,  Limited 
1912 


£*P6  o  1 


Copyright  191 2 
By  Otto  Cohnheim 


PRESS    OF    THE    PUBLISHERS    PRINTING    COMPANY,    NEW    YORK,    U.    S.    A. 


PREFACE 

The  following  lectures  were  given  under  the  Herter 
Foundation  at  the  University  and  Belle vue  Hospital 
Medical  College  in  the  City  of  New  York  in  1910. 

They  were  delivered  before  an  audience  of  physicians 
and  medical  students  and  the  subject  was  treated  from 
the  biological  point  of  view.  Here  let  me  take  occasion 
to  express  my  indebtedness  to  the  members  of  the 
faculty  of  the  College,  as  well  as  to  many  other  colleagues 
in  New  York,  for  their  kindness  during  a  visit  which 
I  shall  ever  remember  with  pleasure. 

I  am  also  indebted  to  Dr.  W.  B.  Cannon  for  assistance 
which  has  enabled  me  to  deliver  these  lectures  in  a 
language  foreign  to  me. 

OTTO   COHNHEIM. 

Heidelberg,  September,  1910. 


COLL 

NEW  V 


CONTENTS 


Preface 
Introduction 


Chapter 

I.  Methods  of  Obtaining  Enzymes 
II.  The  Purification  of  Enzymes 

III.  The  General   Properties  of  Enzymes 

IV.  Enzymes  as  Catalyzers 
V.  The    Reversible    Action    of    Enzymes 

VI.  Enzymes   and    Optical    Activity 
VII.  Mode  of  Action  of  Enzymes 
VIII.  Antiferments 
IX.  Specificity  of  Enzymes 
X.  Zymogens  and  Activators  . 
XI.  The  Individual  Enzymes 
XII.  The  Lipases  or  Steapsins  of  the  Alimentary  Canal 

XIII.  Proteolytic   Enzymes 

XIV.  Miscellaneous  and  Vegetable  Enzymes 

XV.  The   Hydrolytic   Enzymes   of   Tissues,   or 
Enzymes  ..... 

XVI.  Proteolytic  Enzymes  of  Blood 
XVII.  Proteolytic  Enzymes  of  Tissues 
XVIII.  Other  Hydrolytic  Enzymes  of  the  Blood 
XIX.  Urease  and  Nucleases 
XX.  The  Oxidases      ..... 
XXI.  The   Metabolism-Enzymes 
XXII.  The  Fibrin-Ferments 


Index  of  Authors 
General  Index 


Autolytic 


nd  Tissue 


PAGE 

iii 
vii 


I 
II 

20 
31 

39 
44 

55 

63 
66 

67 

73 
81 

84 

99 

108 
110 

115 
119 
121 
124 

145 
162 

167 
171 


INTRODUCTION 

The  term  ''ferments"  was  first  used  early  in  the  nine- 
teenth century  in  the  time  of  Berzelius  and  Schwann. 
Subsequently,  the  ferments  as  chemical  bodies  became 
confused  with  the  micro-organisms  which  cause  fermenta- 
tion. To  avoid  confusion,  Kiihne  gave  the  new  name, 
"  enzymes,"  to  such  digestive  ferments  as  he  knew — 
pepsin,  trypsin,  steapsin,  and  ptyalin.  To-day  we  know 
that  yeast,  the  "ferment"  of  the  earlier  authors,  is  a  com- 
plete organism,  containing  enzymes  like  other  cells  and 
organisms,  although  we  use  the  terms  "ferments"  and 
1 '  enzymes ' '  promiscuously. 

Some  hydrolytis  enzymes  of  the  yeast  and  of  the  digest- 
ive glands  have  been  known  for  a  long  time,  but  interest 
in  the  enzymes  has  grown  in  recent  years,  especially  since 
the  great  discovery  of  zymase  by  Buchner.  It  is  now  the 
general  opinion  of  physiologists  that  living  organisms 
effect  most  chemical  operations  by  means  of  enzymes — 
i.e.,  enzymes  are  the  tools  of  the  cell. 

Earlier  authors  used  for  enzymes  or  ferments  names 
ending  in  "in"  like  pepsin,  trypsin,  ptyalin.  Recently 
most  investigators  have  adopted  names  ending  in  "  ase" 
which  were  suggested  by  French  biologists,  like  maltase, 
lactase,  protease;  and  they  have  tried  to  introduce  a 
rational  nomenclature,  in  which  the  name  shows  the 
action  of  the  enzyme.     The  name  of  the  chemical  com- 

vii 


vill  INTRODUCTION 

pound  which  is  acted  upon  by  the  enzyme  is  taken,  and 
is  connected  with  the  ending  "ase."  Thus  sucrase  is  an 
enzyme  which  acts  upon  sucrose  or  cane  sugar;  protease 
acts  upon  proteins;  and  maltase  upon  maltose.  I  think 
it  will  be  impossible  to  employ  these  designations  very 
closely,  because  proteins  can  be  dissociated  by  different 
enzymes,  e.g.,  peptones  by  two  enzymes  with  different 
qualities.  Glucose  is  converted  by  zymase  into  carbon 
dioxide  and  alcohol,  and  by  the  enzyme  of  the  Bacillus 
acidi  lactici  into  lactic  acid.  Different  qualities  necessitate 
different  names.  I  shall  use  the  historical  names,  and 
employ  the  name  that  was  given  to  the  enzyme  by  its  first 
discoverer,  or  that  which  has  been  generally  adopted. 
Similarly  the  so-called  rational  nomenclature,  as  adopted 
by  all  chemists,  employs  really  the  old  names.  I  shall 
therefore  employ  the  terms  pepsin,  trypsin,  crepsin, 
ptyalin,  invertin,  maltase,  lactase,  zymase,  and  steapsin. 
In  the  case  of  the  enzyme  that  converts  starch  into  dex- 
trin and  maltose,  both  names,  diastase  and  maltase,  are 
authorized  because  the  existence  of  two  enzymes  in  malt 
and  in  saliva  has  been  suggested.  The  methods  which 
enable  us  to  obtain  and  to  work  with  enzymes,  have 
first  to  be  dealt  with,  and  later  comes  the  discussion 
of  the  general  properties  of  enzymes,  after  which  the 
individual  enzymes  will  be  considered. 


ENZYMES 


CHAPTER   I 

Methods  of  Obtaining  Enzymes 

Chemistry  cannot  produce  enzymes.  They  are  found 
only  as  products  of  the  living  protoplasm  of  cells,  and  in 
all  investigations  on  enzymes  they  must  be  separated  from 
the  cells  and  the  organs.  So  far  as  ease  of  separation  is 
concerned,  a  great  difference  exists  between  enzymes — 
the  extracellular  enzymes,  that  act  outside  of  the  cell, 
and  the  intracellular  enzymes,  or  endo-enzymes,  which 
in  living  organisms  never  leave  the  cell. 

The  enzymes  of  the  first  class  are  secreted  by  the 
glands,  and  in  many  cases  these  secretions  can  be 
obtained,  and  with  them  the  enzymes.  Human  saliva  is 
easily  obtained;  and  following  the  methods  of  Pawlow1 
and  of  Bayliss  and  Starling2  we  can  get  pure  saliva, 
pure  gastric  and  pancreatic  juice  of  the  dog,  cat,  horse, 
goat,  and  other  higher  animals.     The  pure  secretions  of 

1  J.  P.  Pawlow:  "Arbeit  der  Verdauungsdnisen,"  1898. — Nagel's 
"Handbuch,"  Bd.  ii.,  1906. 

2  W.  M.  Bayliss  and  E.  H.  Starling:    Journal  of  Physiology,  28  and  29 

(i9°3)« 

I 


2  ENZYMES 

the  digestive  glands  of  some  invertebrates  can  also  be 
obtained.  One  advantage  of  these  methods  is  that  the 
product  is  free  from  many  undesired  substances;  the 
chief  advantage  is  the  certainty  of  having  the  correct 
solubility  and  the  proper  reaction.  Where  the  actual 
secretions  are  obtainable,  investigations  should  never  be 
made  with  extracts. 

In  many  cases,  however,  the  secretions  cannot  be 
obtained.  For  instance,  the  small  intestine  secretes  a 
litre,  or  more,  of  fluid,  but  the  greatest  part  of  this  enteric 
juice  is  reabsorbed  before  running  out  of  a  fistula,  and 
we  can  get  but  a  few  cubic  centimetres  from  a  large  dog, 
or  even  from  the  large  ruminants.  From  small  animals, 
most  invertebrates,  and  micro-organisms,  it  is  naturally 
impossible  to  get  secretions  in  quantities  sufficient  for 
any  precise  investigation.  We  are  forced  to  make  ex- 
tracts from  the  organs  or  from  the  whole  organism,  and 
to  examine  the  enzymes  present  in  such  extracts.  The 
only  fluid  that  dissolves  enzymes  is  water;  no  enzyme  is 
extractable  by  strong  alcohol,  ether,  benzene,  chloroform, 
strong  glycerin,  etc.  Diluted  glycerin  was  often  used  by 
the  earlier  investigators  (Wittich).  It  has  the  advantage 
that  the  growth  of  bacteria  is  checked  by  a  glycerin  of 
seventy  or  eighty  per  cent,  and  that  the  changes  which 
enzymes  undergo  in  watery  solutions  are  prevented  or 
much  retarded  by  glycerin.  Thus  a  solution  of  pepsin 
or  of  trypsin  in  diluted  glycerin  can  be  preserved  for  many 
months  or  even  years,  and  the  quantity  of  the  dissolved 
enzyme  can  be  measured  better  than  if  the  ferment  were 


METHODS    OF   OBTAINING   ENZYMES  3 

in  a  solid  state.  The  enzyme,  however,  is  not  dissolved 
in  the  glycerin  itself,  but  in  the  water  diluting  the  glycerin; 
therefore  diluted  glycerin  dissolves  merely  a  small  quan- 
tity of  the  enzyme,  and  the  glycerin  solutions  of  enzymes 
are  weak  solutions.  If  we  want  a  solution  comparable 
with  the  natural  secreted  juices,  we  must  dissolve  the 
organs  in  water,  but  we  have  the  choice  of  pure  water,  or 
a  salt  solution,  or  a  weak  acid  or  alkaline  solution.  On 
account  of  its  low  osmotic  pressure,  distilled  water 
destroys  the  cells  and  thus  makes  ferments  accessible, 
while  weak  salt  solutions  dissolve  more  proteins  than  does 
pure  water.  For  enzymes,  however,  I  have  seen  no  dif- 
ference between  water  and  salt  solutions.  Acid  solutions 
are  good  for  enzymes  secreted  in  association  with  acid, 
such  as  pepsin,  some  proteolytic  enzymes  of  the  blood 
corpuscles  and  of  invertebrates,  and  also  the  enzymes  of 
yeast.  The  other  enzymes  are  not  destroyed  by  very 
weak  acids,  but  some  cell  proteins  are  precipitated,  and 
many  enzymes  are  absorbed  in  these  proteins  and  pre- 
cipitated with  them.  I  shall  discuss  this  in  treating  of 
the  purification  of  enzymes.  If  there  is  no  special  reason 
for  the  use  of  acids,  as  in  the  case  of  pepsin,  perhaps  the 
best  and  simplest  method  is  to  extract  tissues  with  water, 
and  to  avoid  other  solvents.  It  is  only  with  tissues  which 
become  acid  immediately  after  death,  as  in  the  case  of  the 
pancreas  and  the  muscles,  when  oxygen  is  lacking,  that 
it  may  be  advantageous  to  use  a  weak  solution  of  sodium 
carbonate  similar  in  its  reaction  to  that  of  most  living 
tissues.     A    stronger    alkaline    reaction   endangers    most 


4  ENZYMES 

enzymes,  and  must  be  avoided.  With  water,  or  a  very 
weak  alkaline  solution,  it  is  very  easy  to  extract  all 
enzymes  destined  for  secretion.  I  have  obtained  extracts 
of  the  stomach  and  of  the  pancreas  containing  more  pepsin 
or  trypsin  than  the  natural  juices.  I  have  never  seen 
peptone  so  rapidly  split  by  the  enteric  juice  as  by  a  watery 
extract  of  the  intestinal  mucous  membrane.  The  ex- 
traction is  more  nearly  complete  if  the  glands  or  the 
mucous  membranes  are  finely  minced  and  ground  with 
sand.  In  this  way,  I  have  extracted  from  the  intestinal 
mucous  membrane  all  the  erepsin  in  thirty  minutes,  using 
three  portions  of  water.  It  is  also  very  easy  to  extract 
diastase,  maltase,  and  invertin  from  yeast,  or  the  ptyalin 
from  the  salivary  glands,  in  a  short  time. 

More  difficulty  is  experienced  in  extracting  the  endo- 
enzymes  from  the  organs.  The  submaxillary  gland  and 
the  liver  contain  the  same  two  enzymes,  diastase  or  ptyalin, 
which  split  starch  or  glycogen  into  maltose,  and  maltase, 
which  changes  maltose  into  glucose. 

There  does  not  seem  to  be  any  great  difference  between 
the  salivary  gland  and  the  liver  in  the  quantity  of  the  two 
enzymes  contained  in  the  glands.  A  great  deal  of  glycogen 
is  lost  if  the  enzymes  of  the  liver  are  not  destroyed  im- 
mediately after  death;  i.e.,  the  liver  yields  much  diastase 
and  maltase.  The  enzymes  cannot  be  extracted  with 
equal  ease  from  the  salivary  gland  and  liver. 

In  1863  my  father1  obtained  from  the  salivary  gland  a 

1  J.  Cohnheim:    Virchow's  Archiv,  28,  241  (1863). 


METHODS   OF   OBTAINING   ENZYMES  5 

solution  splitting  starch  in  one  minute;  and  using  the 
same  method,  he  obtained  from  the  liver  a  solution  splitting 
starch  in  two  hours.  Forty  years  later,  Borchardt 1  had 
no  better  results  when  isolating  the  ptyalin  of  the  liver. 
Neither  from  yeast  nor  from  the  muscles  can  the  glycolytic 
enzymes  be  extracted  without  complete  disintegration  of 
cells  and  cell  structure.  For  fifty  years  biologists  debated 
whether  the  fermentation  of  sugar  by  yeast  was  an  en- 
zymotic  process,  or  whether  only  the  living  yeast  could 
burn  sugar.  This  was  decided  when  E.  and  H.  Buchner 
and  Hahn 2  were  able  to  isolate  the  zymase  from  the  cell 
after  thoroughly  destroying  the  cell  membranes.  The 
chemical  means  for  dissolving  out  substances  like  glyco- 
gen from  the  tissues,  using  strong  sodium  hydroxide  or 
other  strong  alkalies,  cannot  be  used,  because  they  de- 
stroy the  enzymes.  The  disintegration  must  be  made 
by  mechanical  means.  Buchner  and  many  others 
have  ground  micro-organisms  and  tissues  with  sand 
and  siliceous  earth,  and  subjected  the  mass  to  consid- 
erable pressure  in  a  hydraulic  press.  Macfadyen  and 
Rowland  have  frozen  yeast  at  a  temperature  of  — 1800 
C,  and  ground  the  solid  ice  without  any  addition.  For 
muscles,  glands,  and  other  tissues,  the  best  method  is 
KosseFs.3  The  tissues  or  the  whole  animal  are  frozen 
by  solid  carbon  dioxide,  or  by  a  salt-and-ice  freezing  mix- 

1  L.  Borchardt:    Pfluger's  Archiv,  ioo,  259  (1903). 

2  E.  Buchner,  H.  Buchner,  and  M.  Hahn:     "Die  Zymasegarung," 
Munich,  1903. 

3  Berichte  d.  d.  chem.  Ges.,  7>2>  (1900). 


6  ENZYMES 

ture,  and  cut  by  rotating  knives.  In  this  manner  a  snow 
is  obtained  which  has  no  visible  microscopical  structure; 
and  this  is  mixed  with  siliceous  earth  and  subjected  to  the 
high  and  slowly  increasing  pressure  of  a  hydraulic  press. 
Using  this  method,  I  have  obtained  60  cc.  of  a  clear 
liquid  from  100  grammes  of  fresh  muscles  of  the  cat  or 
ox,  and  40  cc.  of  liquid  from  100  grammes  of  liver,  spleen, 
or  kidney.  This  liquid  contains  a  fair  quantity  of  the 
intracellular  enzymes,  but  only  a  small  amount  of  the  cell 
proteins.  There  is  no  certainty  that  we  have  in  solution 
all  the  enzymes  existing  in  the  cells  or  organs.  Recently 
Pringsheim  and  Zemplen,1  in  the  laboratory  of  E.  Fischer, 
tried  to  extract  the  sucroclastic  enzymes  from  some 
bacteria,  and  succeeded  in  many  cases,  but  not  in  all, 
although  all  investigated  bacteria  contain  the  enzymes. 
The  enzymes  are  retained  by  the  proteins,  while  the 
other  insoluble  substances  remain  with  the  siliceous  earth. 
We  may  now  proceed  to  discuss  the  difficult  and  im- 
portant question  of  adsorption,  or  attachment  of  enzymes 
to  proteins  and  other  substances.  In  many  cases  we  can 
overcome  the  adsorption  and  render  the  enzymes  soluble, 
if  we  change  the  reaction  of  the  pulp  before  expressing  it. 
Many  enzymes  resemble  the  nucleoproteins  in  solubility, 
and  are  precipitated  by  very  weak  acids.  Those  enzymes, 
especially,  that  convert  disaccharides  into  monosaccha- 
rides, as  lactase,  maltase,  and  invertin,  are  fixed  in,  and  re- 
tained by,  the  solids  of  the  tissues.     Another  method  of 

1  "Studien   iiber   die   Polysaccharide   spaltenden   Fermente   in   Pilz- 
presssaften";  Zeitschr.  f.  physiolog.  Chemie,  62  (1909). 


METHODS  OF  OBTAINING  ENZYMES  7 

disintegrating  cells  or  tissues  is  by  drying  them.  Kiihne 
treated  fresh  pancreas  with  alcohol  and  ether  in  great 
excess;  the  well-dried  powder  contained  all  the  enzymes  of 
the  gland.  The  dried  pancreas  powder  was  used  also  by 
Malloizel  in  his  important  investigations  on  the  synthesis 
of  fats  induced  by  the  enzyme  steapsin.  Buchner  placed 
yeast  in  an  excess  of  "  acetone  and  thus  obtained  the 
"dauerhefe,"  i.e.,  a  dry  powder  containing  zymase. 
Wiechowski  dissolved  the  fats  and  lipoids  of  the  fresh 
liver  in  toluene,  and  dried  the  undissolved  residue  at  500  C. 
The  enzymes  can  be  preserved  for  a  long  time  as  dry 
powder,  but  they  lose  their  solubility.  The  " dauerhefe" 
when  added  to  a  sugar  solution  ferments  the  sugar,  but 
it  is  not  possible  to  extract  the  zymase  from  this  prepara- 
tion. It  is  generally  necessary  to  purify  the  enzymes  and 
this  is  done  by  removing  contaminating  substances  from 
the  fresh  extracts  and  then  evaporating  the  pure  solution 
to  dryness.  The  pepsin  and  trypsin  of  commerce  are 
dry  powders  prepared  in  this  way. 

Between  the  endo-enzymes  working  within  the  proto- 
plasm and  the  enzymes  destined  for  secretion,  are  the 
enzymes  of  the  mucous  membranes  of  the  stomach  and  of 
the  intestine  in  which  the  chyme  is  absorbed.  The  chyme 
passes  through  the  cells,  but  becomes  no  part  of  the 
protoplasm.  It  has  been  observed  that  the  enzymes 
formed  by  the  cells  of  the  small  intestine — the  erepsin, 
the  invertin,  the  maltase,  and  especially  the  lactase — are 
to  be  found  in  very  large  quantity  in  the  extract  of  the 
mucous  membrane,  but  in  small  quantity  in  the  secretion 


8  ENZYMES 

or  in  the  contents  of  the  intestine.  It  seems  to  me  that 
the  simplest  explanation  of  this  fact  is  that  the  peptones 
and  the  disaccharides  are  converted  to  a  small  extent  in 
the  lumen  of  the  intestine,  and  to  a  greater  extent  while 
they  pass  through  the  cells.  For  the  stomach  this  explana- 
tion seems  certain.  Tobler,1  when  investigating  the 
digestion  in  the  stomach,  using  a  good  quantitative 
method,  found  that  twenty  to  thirty  per  cent  of  the  proteins 
of  fresh  meat  are  absorbed  in  the  stomach,  particularly  in 
the  right  side  of  the  stomach  in  the  antrum  pylori.  Berg- 
mann 2  has  discovered  a  protease  or  an  erepsin  which  splits 
peptone  into  amino-acids,  in  the  mucous  membrane  of 
the  antrum  pylori,  but  this  protease  is  not  present  in  the 
chyme  ejected  through  the  pylorus;  at  least  I  could  find 
no  traces  of  it.3  The  function  of  this  strong  enzyme  is  to 
attack  peptone  passing  through  the  cells  of  the  absorbing 
membrane.  These  enzymes — the  protease  of  the  stomach 
and  the  erepsin  and  the  lactase  of  the  intestine — work 
within  the  cell,  but  they  can  be  as  easily  extracted  as  those 
enzymes  which  are  secreted. 

Some  authors  have  believed  that  they  could  investigate 
tissue  enzymes  without  dissolving  the  tissues.    Salkowski, 
Jacoby,5  Hedin  and   Rowland,6  and   others  have  placed 

1  L.  Tobler:    Zeitschr.  f.  physiolog.  Chemie,  45  (1905). 

2  P.  Bergmann:    Skandinav.  Archiv  f.  Physiol.,  18  (1906). 
aCohnheim:  Miinchener  med.  Wochenschrift,   1907. 

4  E.  Salkowski:    Zeitschr.  f.  klin.  Medicin,  17  (Suppl.),  1891. 

5  M.  Jacoby:    Zeitschr.  f.  physiolog   Chemie,  30  (1910). 

6  S.  G.  Hedin  and  S.  Rowland:     Zeitschr.  f.    physiolog.    Chemie,  32 
(1901). — O.  Schumm,  Hofmeister's  Beitrage,  7  (1905). 


METHODS    OF    OBTAINING   ENZYMES  9 

finely  minced  liver  in  water  saturated  with  chloroform  or 
toluene,  and  allowed  the  enzymes  of  the  dead  cells  to  act 
on  the  substances  surrounding  them. 

For  some  time  I  followed  the  same  method  in  investigat- 
ing the  glycolytic  enzymes  of  muscles.  I  placed  the  finely 
minced  muscles  in  water  containing  glucose  and  the 
activator  of  the  enzyme  extracted  from  the  pancreas. 
We  can  detect  and  estimate  in  this  manner  unstable 
enzymes  which  are  destroyed  whilst  being  extracted,  but 
we  lose  the  advantage  of  limiting  the  action  to  selected 
substances. 

Many  proteolytic  or  so-called  autolytic  enzymes  of  the 
liver,  spleen,  and  kidney  can  be  extracted  without  difficulty 
in  the  same  manner  as  erepsin  from  the  mucous  mem- 
branes; and  with  the  exception  of  this  extract,  we  know 
only  of  the  existence  in  these  organs  of  some  proteolytic 
enzymes.  We  can  find,  for  instance,  in  the  liver  three 
classes  of  proteolytic  enzymes  of  different  characters  and 
different  biological  importance.  First,  enzymes  derived 
from  the  blood  left  in  the  vessels  of  the  liver.  These 
interesting  enzymes,  which  Opie 1  has  investigated  so 
thoroughly,  will  be  dealt  with  later.  Secondly,  there  is 
erepsin,  destined  to  act  upon  the  peptones  which  have 
passed  the  intestinal  wall  unchanged  and  which  circulate 
in  the  blood.  Thirdly,  there  may  be  an  enzyme  which 
attacks  the  proteins  of  the  living  protoplasm,  and  thus  in 


1  E.  L.  Opie:     Rockefeller  Institute  for  Medical  Research,  4  (1905); 
6(1906);   8(1908). 


10  ENZYMES 

metabolism  draws  upon  the  real  body  of  the  cells.  Ap- 
parently there  would  be  a  great  difference  in  metabolism 
whether  the  enzyme  attacked  absorbed  protein  or  the 
protein  of  the  cell  itself.  If  we  extract  the  enzyme,  we 
can  distinguish  between  erepsin  and  trypsin,  because 
trypsin  leaves  untouched  the  true  albumins  and  globulins 
of  the  protoplasm,  while  erepsin  slowly  attacks  the  histone 
of  the  nucleus.  The  strong  proteolytic  enzyme  yielded 
by  the  so-called  liver  of  molluscs  nearly  resembles  erepsin. 
Vernon  and  Hedin  1  extracted  and  isolated  the  enzymes 
and  demonstrated  their  resemblance  to  erepsin.  The 
study  of  the  enzymes  within  the  tissue,  and  without 
extracting  them,  cannot  be  even  approximately  complete; 
we  must  always  try  to  extract  the  enzymes. 

1  Vernon:  Journ.  of  Physiol.,  30  and  33. 


CHAPTER  II 

The  Purification  of  Enzymes 

For  many  purposes  we  can  use,  without  further  treat- 
ment, the  extracts  of  the  tissues  containing  ferments. 
The  investigations  of  zymase  by  Buchner1  and  his  collabo- 
rators were  made  with  the  press-juice  of  the  yeast.  Emil 
Fischer  has  studied  the  enzymes  by  hydrolyzing  the  disac- 
charides  with  the  simple,  unpurified  extracts  of  yeasts 
and  animal  tissues.  For  the  glycolytic  enzymes  in  the 
tissues  of  the  higher  animals,  no  method  is  known  to-day 
by  which  the  enzymes  may  be  purified  without  destroying 
them.  Other  enzymes  withstand  chemical  treatment, 
and  in  many  investigations  it  is  necessary  to  remove 
proteins,  nucleic  acids,  salts,  carbohydrates,  etc.,  accom- 
panying the  enzymes  in  the  watery  extract.  Even  the 
natural  secreted  juices  are  not  pure  solutions  of  enzymes. 
The  saliva  and  the  enteric  juice  contain  sodium  chloride 
and  sodium  carbonate;  the  pancreatic  juice  and  the 
secretion  of  the  liver  of  the  cephalopods  contain  sodium 
carbonate  and  perhaps  carbonates  of  organic  bases;  the 
gastric  juice  contains  hydrochloric  acid;  and,  in  addition, 
all  these  juices  contain  proteins  and  nucleic  acids,  and 
perhaps  also  lecithins.     The  organism  can  secrete  liquids 

1  E.  and  H.  Buchner:  "Die  Zymasegarung,"  Munich,  1903. — E. 
Buchner  and  his  collaborators:    Ber.  d.  deutsch.  chem.  Ges.,  1896-1908. 

II 


12  ENZYMES 

which  do  not  contain  proteins  or  nucleic  acids,  e.g., 
urine,  bile,  sweat;  or  liquids  which  contain  these  bodies 
in  traces,  e.g.,  the  aqueous  humor,  the  cerebrospinal 
fluid,  and  some  liquids  in  invertebrates,  for  instance 
echinodermata.  But  these  secretions  are  free  from 
enzymes.  The  secretions  containing  enzymes  are  rich 
in  proteins  and  nucleic  acids.  The  quantity  of  these 
in  the  gastric  and  pancreatic  juice  varies  proportionately 
with  the  enzymes,  and  their  solubility  is  like  that  of  the 
enzymes.  Therefore  many  authors  have  thought  them 
to  be  the  enzymes;  but  they  are  mistaken,  because  we 
can  separate  the  enzymes  from  the  proteins  accompany- 
ing them  in  extracts  and  in  secretions.  I  shall  discuss 
their  chemical  nature  later  on. 

In  order  to  purify  substances  chemically,  we  precipitate 
either  the  substance  or  the  impurities.  Now  we  do  not 
know  any  reagent  which  will  bring  down  enzymes  in  a 
specific  manner,  but  the  latter  have  the  property  of  being 
adsorbed  by  finely  divided  solids,  and  especially  by 
precipitates  during  their  formation  in  a  solution.  We 
have  to  consider  three  possibilities,  although  practically 
it  is  not  easy  to  tell  which  one  we  are  dealing  with  in  a 
special  case. 

i.  Enzymes  and  other  substances  are  similar  to  one 
another  in  solubility  and  precipitability.  Thus  all  pro- 
teins occurring  in  animal  or  vegetable  bodies  are  salted 
out  by  ammonium  sulphate,  and,  so  far  as  we  know,  so 
are  all  enzymes. 

2.  Enzymes  are  brought  down  by  physical  adsorption, 


THE   PURIFICATION   OF   ENZYMES  13 

by  precipitates  forming  in  an  enzyme  solution;  for  in- 
stance, by  the  precipitate  of  calcium  phosphate  formed 
on  adding  lime-water  and  phosphoric  acid,  or  the  pre- 
cipitate of  cholesterin  formed  on  adding  an  alcoholic 
solution  of  cholesterin,  to  the  aqueous  solution  of  enzymes. 
Proteins,  animal  charcoal,  and  siliceous  earth  also  bind 
enzymes  by  physical  adsorption. 

3.  Enzymes  are  fixed  and  held  by  solid  substances  in 
chemical  connection,  and  in  this  manner  they  are  attached 
to  many  proteins,  in  addition  to  the  mere  physical  adsorp- 
tion, which  occurs  in  the  cases  of  animal  charcoal  and 
siliceous  earth.  Physical  adsorption  precipitates  all  en- 
zymes, while  the  chemical  connection  is  a  specific  one; 
but  the  connection  is  apparently  a  loose  one  which  can  be 
broken  by  hydrolysis.  The  completeness  of  hydrolytic 
dissociation  depends  on  the  amount  of  water,  and  we  can 
thus  separate  enzymes  and  proteins,  or  enzymes  and  animal 
charcoal,  by  the  addition  of  an  excess  of  water;  we  can 
wash  out  the  enzymes  from  the  solids,  though  they  are 
chemically  connected  with  them. 

The  chemical  connection,  the  physical  adsorption,  and 
the  precipitability  of  enzymes  in  the  presence  or  absence 
of  proteins,  are  available  for  the  purification  and  separa- 
tion of  enzymes  chiefly  by  three  methods : 

1.  We  can  precipitate  both  enzymes  and  proteins  with 
alcohol  (Vernon1),  with   ammonium  sulphate    (Kiihne 2 


1  H.  M.  Vernon:     Journ.  of  Physiology,  30  (1903). 

2W.  Kiihne:     Naturhist.  Med.  Verein,  Heidelberg,  3  (1886). 


14  ENZYMES 

and  others),  with  uranyl  acetate  (Jacoby  1 — the  alde- 
hydase  of  the  liver;  Roselle2 — trypsin),  or  with  phos- 
phoric acid  and  lime-water  (Brucke  and  others — 
gastric  juice  and  saliva).  We  allow  the  precipitates  to 
stand  for  some  time,  until  the  proteins  become  insoluble, 
and  then  dissolve  out  the  enzyme  with  water,  or  in  the 
case  of  pepsin,  with  hydrochloric  acid.  Using  the  phos- 
phoric acid  and  lime-water  method,  Brucke  isolated 
pepsin,  though  with  great  loss,  as  the  first  enzyme  to  be 
obtained  at  all  free  from  proteins;  my  father  employed 
the  same  method  for  ptyalin,  which  was  not  destroyed 
by  this  treatment.  So  with  ammonium  sulphate  the 
erepsin  of  the  intestinal  mucous  membrane  was  separated 
from  a  great  mass  of  the  proteins  and  other  impurities. 
The  ammonium  sulphate  and  the  other  crystalloids  were 
subsequently  removed  by  dialysis,  leaving  a  solution  rich 
in  ferments  but  poor  in  all  other  substances. 

2.  We  can  split  the  proteins  wholly  into  amino-acids  or 
peptones,  and  then  precipitate  the  enzyme  with  ammoni- 
um sulphate.  By  this  method  Kuhne  obtained  nearly 
pure  trypsin.  This  method  is  used  for  pepsin  and  for  the 
enzyme  oxidizing  aldehydes  in  the  liver,  but  cannot  be 
applied  to  most  enzymes  which  are  destroyed  by  strong 
proteolytic  ferments.  Even  trypsin  itself,  purified  in 
this  manner,  loses  a  great  deal  of  its  power. 

3.  We  can  precipitate  many  proteins,  nucleic  acids,  and 
other  substances,  by   very  dilute  acetic  acid,  while  the 

1  M.  Jacoby:    Zeitschr.  f.  physiol.  Chemie,  3  (1900). 

2  Roselle:    Diss.  Strassburg,  1901. 


THE   PURIFICATION    OF   ENZYMES  15 

enzyme  remains  in  solution.  In  such  manner  Kossel  and 
Dakin i  freed  liver  arginase  from  most  proteins.  Essentially 
the  same  principle  applies  when  fresh  tissue-extracts  are  al- 
lowed to  stand  for  some  time;  coagulation  being  then  due 
to  the  weak  acid  reaction  resulting  in  muscles  and  other 
tissues  shortly  after  death.  If  we  dialyze  the  extract  at 
this  time  in  running  water,  we  remove  both  the  proteins 
which  become  insoluble  and  the  salts  and  other  crystalloid 
substances  which  pass  through  the  parchment.  After 
the  dialysis,  or  after  standing,  we  filter  off  the  precipitate. 
As  a  rule  it  is  useless  to  filter  under  pressure  through  a 
Chamberland  or  similar  filter. 

Nevertheless,  the  advantage  of  Buchner's  method  con- 
sists not  only  in  the  complete  disintegration  of  tissues  and 
structures,  but  likewise  in  the  filtration  of  the  press-juice 
through  siliceous  earth,  which  retains  the  nucleoproteins, 
the  globulins,  and  all  proteins  coagulating  spontaneously. 
Almost  the  only  proteins  found  in  the  press-juice  of  the 
muscles  and  the  liver  are  albumin  and  the  haemoglobin  of 
the  blood.  These  juices  are  in  most  cases  very  rich  in 
enzymes. 

Using  one  of  these  methods  it  is  easy  to  obtain  pro- 
teolytic enzymes  and  steapsins,  but  many  sucroclastic 
enzymes  are  retained  by  the  proteins,  and  cannot  be 
dissolved  out  again  by  water.  Sometimes  it  is  possible 
to  dissolve  them  by  very  weak  alkali,  but  the  necessary 
conditions  have  not  been  sufficiently  studied. 

1  A.  Kossel  and  H.  D.  Dakin:  Zeitschr.  f.  physiol.  Chemie,  41,  42 
(1904). 


16  ENZYMES 

If  we  do  not  succeed  in  bringing  the  enzymes  into  solu- 
tion, we  must  add  the  solid  precipitate  to  the  solution 
containing  carbohydrates  or  fats  or  proteins,  which  are 
to  be  acted  upon  by  the  enzyme.  We  do  not  know  whether 
the  enzyme  works  under  such  conditions  in  a  solid  state 
and  converts  carbohydrates  and  other  substances  only 
by  contact,  or  whether  the  enzyme  passes  into  solution. 
We  shall  see  that  we  must  assume  a  chemical  connection 
and  combination  between  enzyme  and  substance.  Per- 
haps the  chemical  compound  thus  newly  formed  is  more 
soluble  than  the  free  enzyme.  This  would  be  a  good  ex- 
planation of  the  action,  for  instance,  by  which  the  mucous 
membrane  of  the  small  intestine  converts  milk  sugar, 
although  it  is  impossible  to  find  lactase  in  the  extracts, 
or  the  ability  of  dried  pancreas  powder  to  dissolve  protein, 
which  is  lacking  in  extracts  made  from  this  powder. 
We  must  always  remember,  however,  that  the  difference 
can  be  produced  also  by  an  improper  reaction  of  the 
extract.  It  is  an  important  fact,  however,  that  we  can 
use  the  dry  powder  for  many  purposes. 

With  these  methods  we  can  easily  remove  proteins  and 
other  harmless,  indifferent  substances.  The  chief  diffi- 
culties which  complicate  the  purification,  extraction,  and 
investigation  of  enzymes,  are  the  presence  of  other 
enzymes,  and  the  anchorage  of  the  enzymes  in  the  cells. 
The  avoidance  of  these  difficulties  is  the  chief  advantage 
of  the  secreted  natural  juices  as  compared  with  the 
extracts. 

i.  By  no  method  can  one  enzyme  be  separated  from 


THE   PURIFICATION   OF   ENZYMES  17 

another  without  great  loss.  The  gastric  juice  contains 
only  the  well-known  proteolytic  enzyme,  pepsin,  which 
dissolves  natural  proteins  and  converts  them  into  pep- 
tones; but  the  process  never  goes  beyond  the  stage  of 
yielding  biuret-giving  substances.  The  mucous  mem- 
brane of  the  stomach,  and  therefore  its  extracts,  contains, 
besides  pepsin  destined  for  secretion,  an  erepsin  acting 
intracellularly  and  splitting  peptones  into  amino-acids. 
The  pepsin  and  the  erepsin  have  the  same  solubilities, 
and  if  we  free  the  proteolytic  enzyme  from  impurities,  we 
concentrate  both  enzymes,  getting  an  artificial  juice  with 
properties  other  than  those  of  the  natural  one.  The  con- 
tradictions of  authors  on  the  question  of  trypsin  and 
trypsinogen  is  believed  by  Bayliss  *  to  be  susceptible  of 
the  same  explanation. 

2.  If  we  microscopically  study  the  pancreas  or  a 
salivary  gland  at  rest,  we  see  the  cell  crowded  with  small, 
refractive  granules  forming  a  mass  that  leaves  only  a 
very  narrow  clear  space  next  to  the  basement  membrane. 
In  the  salivary  gland  the  cell  can  be  wholly  studded  with 
the  granular  mass.  During  glandular  activity  the  granules 
become  much  fewer  in  number,  and  retract  to  an  inner 
narrow  margin;  in  the  salivary  gland  they  may  even  dis- 
appear. There  can  be  no  doubt  that  during  the  act  of 
secretion,  the  granules  are  discharged  to  form  part  of  the 
secretion.  They  become  the  solid  matter  of  the  juices; 
therefore  they  must  be  soluble  in  water.     If  we  extract 

1  W.  M.  Bayliss:  Archiv.  des  Sciences  Biol,  de  St.  Petersbourg,  n 
(i  Suppl.),  1901. 

2 


18  ENZYMES 

the  pancreas  or  the  parotid  gland  while  studded  with  these 
granules,  they,  and  with  them  the  enzymes,  are  dissolved 
immediately.  As  has  been  pointed  out  by  Pekelharing,1 
we  find  in  the  extracts  of  the  gastric  mucous  membrane 
the  most  characteristic  chemical  compound  of  the  gastric 
juice. 

There  is  something  else  to  be  noticed  in  dealing  with 
the  intracellular  enzymes  that  never  leave  the  cell.  In 
the  state  of  activity,  both  these  enzymes  and  the  sub- 
stances hydrolyzed  or  oxidized  by  them  must  be  in 
solution,  for  "  corpora  non  agunt  nisi  soluta."  To  avoid 
the  action  of  enzymes  at  rest,  the  two  bodies  must  be 
separated  from  each  other;  they  must  be  fixed  outside 
the  real  fluid  protoplasm.  We  know  another  substance 
which,  in  this  respect,  is  analogous  to  the  endo-enzymes; 
glycogen,  which  is  soluble  in  water,  but  which  may  be 
stored  in  insoluble  form  within  the  cell.  Ehrlich 2  found 
that  glycogen  is  fixed  in  the  liver  cell  by  a  "Trager- 
substanz,"  a  holding-substance,  and  that  one  of  the  color 
reactions  attributed  to  glycogen  is  due  to  this  substance. 

Glycogen  and  this  Tragersubstanz  are  rather  firmly 
bound  together.  We  know  with  what  difficulty  the 
glycogen  is  completely  dissolved  out  of  the  organs,  and 
with  what  difficulty  it  is  freed  from  the  last  traces  of  im- 
purities. It  seems  to  me  that  we  must  also  assume  such 
a  holding-substance  for  the  intracellular  enzymes  which 
restrains  the  action  of  the  enzyme  even  after  the  almost 

1  Pekelharing:    Zeitschr.  f.  physiol.  Chemie,  22  (1896);  38  (1902). 

2  P.  Ehrlich:    in  a  paper  in  French's  Zeitschr.  f.  klin.  Med.,  6  (1883). 


THE   PURIFICATION   OF   ENZYMES  19 

complete  disintegration  of  the  tissues.  I  have  cut  frozen 
muscles  using  Kossel's *  rotating  knives,  and  I  have 
studied  the  glycolytic  enzyme  of  this  snow;  I  have  also 
compared  the  snow  simply  dissolved  and  floating  in 
water,  with  the  press-juice  I  obtained  by  mixing  the  snow 
with  siliceous  earth  and  pressing  it.  The  glycolytic 
power  of  the  press- juice  was  much  greater,  I  think,  be- 
cause the  siliceous  earth  had  retained  with  the  other 
proteins  the  Tragersubstanz  of  the  enzyme. 

1  Kossel:    Zeitschr.  f.  physiol.  Chemie,  33  (1901). 


NEW  VG 


CHAPTER  III 

The  General  Properties  of  Enzymes 

All  enzymes  are  colloids,  like  the  native  proteins  of 
the  seralbumin  type.  Like  these,  they  cannot  be 
dialyzed  through  parchment,  and  are  irrevocably  changed 
on  heating  in  aqueous  solution  to  6o°  to  700  C.  The 
albumin  is  coagulated  by  heat,  and  this  coagulum  cannot 
be  dissolved  without  conversion  into  peptones;  the 
enzymes,  however,  disappear  entirely  on  heating.  It  is 
possible  that  they  are  coagulated  and  become  insoluble; 
it  is  also  possible  that  they  are  chemically  changed  and 
decomposed.  The  only  sign  of  the  presence  of  enzymes 
is  their  action,  and  we  see  that  the  hydrolyzing  or 
oxidizing  power  of  a  solution  is  lost  by  heating,  and  can- 
not be  restored.  Pfeffer  was  able  to  show  that  the 
enzymes  do  not  volatilize  with  the  vapor  of  water  and 
thus  do  not  leave  the  solution. 

All  enzymes  are  destroyed  by  heat.  Most  of  them 
are  more  or  less  slowly  impaired  at  a  low  temperature — 
370  to  400  C. — and  many  undergo  a  slow  loss  of  power, 
at  room  temperature,  and  even  at  zero.  Zymase  and  the 
glycolytic  enzyme  of  muscles  are  destroyed  in  the  frozen 
state  in  two  to  four  days.1  Enzymes  are  also  broken 
down  by  many  chemical  processes,  by  strong  acids  and 

1  L.  Iwanoff:    Zeitschr.  f.  physiol.  Chemie,  42  (1904). 

20 


THE  GENERAL  PROPERTIES  OF  ENZYMES     21 

alkalies,  by  alcohol,  etc.  Pepsin  loses  its  enzymotic 
properties  by  contact  with  the  weakest  alkali,  and  many 
oxidizing  enzymes  by  acid.  It  is  possible  that  this 
alteration  is  the  same  as  that  which  takes  place  at  a  slower 
rate  on  heating.  Bayliss l  supposes  that  the  alteration 
at  low  temperature  is  reversible.  He  cites  as  an  example, 
that  toxins  may  become  inactive  toxoids  while  retaining 
their  chemical  character.  This  supposition  was  re- 
cently supported  in  a  very  remarkable  manner  by  Pawlow.2 
It  has  always  been  believed  that  the  pepsin  is  completely 
destroyed  by  alkali;  Pawlow  and  Tichomirow  demonstrat- 
ed that  this  is  the  case  only  if  the  alkaline  solution  is  im- 
mediately acidified;  the  pepsin  cannot  then  be  detected. 
If,  however,  the  gastric  juice  be  made  alkaline,  then 
neutralized,  and  allowed  to  stand  for  some  time  before 
acidulating  it,  the  pepsin  does  not  disappear,  but  acts 
almost  as  strongly  as  before.  This  is  the  first  case  of 
restitution  of  an  enzyme  after  having  lost  its  vigor.  Melt- 
zer  has  pointed  out  that  this  slow  loss  of  power  is  greatly 
accelerated  on  shaking  solutions  of  enzymes.  The  de- 
struction through  shaking  takes  place  even  at  low  tem- 
peratures, but  proceeds  more  rapidly  at  the  temperature 
of  the  body. 

To-day  every  investigation  of  enzymes  is  retarded  by 
the  same  difficulties  as  those  attending  the  chemical  study 
of  proteins.     The  true  proteins  are  like  enzymes  so  far  as 

1  W.  M.  Bayliss:  Archiv.  des  Sciences  Biologiques  de  St.  Petersbourg, 
ii  (Suppl.  Pawlow-Volume)  (1904). 

2  N.  P.  Tichomirow:   Zeitschr.  f.  physiolog.  Chemie,  55   (1908). 


22  ENZYMES 

sensitiveness  toward  temperature  and  toward  acid  and 
alkali  are  concerned.  Knowledge  of  the  chemistry  of  the 
proteins  stagnated  for  a  long  time,  because  the  methods  of 
dealing  with  colloids  were  undeveloped.  It  made  very 
rapid  progress  as  soon  as  it  was  found  possible  to  work 
with  strong  hydrochloric  acid,  barium  hydroxide,  and 
phosphotungstic  acid.  I  have  cited  the  methods  of 
purifying  enzymes,  salting  out,  and  precipitating  by  weak 
acid  or  alcohol,  avoiding  heat.  By  such  methods  we  are 
unable  to  separate  compounds  which  can  be  chemically 
defined.  It  is  to  be  hoped  that  Pawlow's  observation 
may  become  the  beginning  of  a  really  chemical  explora- 
tion of  enzymes. 

We  cannot  detect  or  estimate  the  enzymes  themselves. 
We  can  only  observe  their  action.  For  example,  we  can 
estimate  the  quantity  of  cane  sugar  before  and  after  the  ac- 
tion of  invertin;  we  can  also  determine  the  quantity  of  glu- 
cose and  levulose,  the  products  of  the  decomposition  of  the 
cane  sugar.  Further,  we  can  estimate  the  quantity  of  the 
coagulable  proteins  before  the  action,  and  after  five,  ten, 
and  fifteen  minutes,  or  the  quantity  of  the  non-coagulable 
peptone;  but  we  cannot  directly  estimate  the  invertin  or 
pepsin.  Therefore  everything  that  disturbs  the  action 
of  the  enzyme  seems  to  be  a  diminution,  while  everything 
that  improves  the  action  seems  to  be  an  increase,  of  the 
quantity  of  the  enzyme,  although  possibly  only  due  to 
differences  in  the  conditions  of  its  action. 

Under  these  circumstances  it  seems  to  me  impossible 
to  speak  of  the  physical  properties  of  enzymes.     It  is  a 


THE  GENERAL  PROPERTIES  OF  ENZYMES     23 

rule,  generally  acknowledged  in  chemistry,  that  for  de- 
termining the  physical  properties  of  a  substance — the 
molecular  weight,  the  melting-point,  the  osmotic  pressure, 
the  rotatory  power,  the  electrical  conductivity,  the  ioniza- 
tion— the  substance  must  be  wholly  purified.  We  know 
that  in  enyzme  solutions  the  weight  of  enzymes  is  much 
smaller  than  the  weight  of  the  impurities;  it  is  clear  that 
in  such  a  solution  we  cannot  define  the  law  of  action  of 
these  bodies.  If  we  read  the  books  or  papers  written  on 
enzymes  by  chemists,  we  find  that  the  law  of  Schultz 
states  that  the  action  of  a  solution  of  an  enzyme  increases, 
not  in  proportion  to  the  quantity,  but  to  the  square  root  of 
the  quantity  of  the  contained  enzyme;  and  the  question  is 
also  debated,  whether  the  enzyme  is  consumed  during  its 
action  or  not,  etc.  The  prevailing  great  interest  in  the 
enzymes  has  led  authors  to  bring  up  questions  which  no- 
body can  answer  at  the  present  time.  All  we  can  do  at 
present  is  to  describe  a  number  of  the  properties  of 
enzymes  which  have  been  observed. 

i.  Enzymes  are  colloids,  and  do  not  dialyze  through 
parchment. 

2.  Enzymes  have  an  optimum  temperature  of  action. 
It  is  well  known  that  all  chemical  processes  go  faster  with 
increasing  temperature,  and  van  't  Hoff  enunciated  the 
rule  that  the  rapidity  of  most  chemical  processes  is  doubled 
or  trebled  when  the  temperature  is  raised  io°  C.  The 
enzymes  follow  this  rule,  but  only  between  o°  and  400  C. ; 
above  400  C,  this  power  decreases  rapidly.  If  we  graphi- 
cally represent  the  behavior  of  the  decomposition  of  cane 


24  ENZYMES 

sugar  by  hydrochloric  acid  and  by  invertin,  with  increasing 
temperature,  we  find  for  the  acid  a  curve  like  this^x^, 
and  for  the  enzyme  a  curve  /^\  ,  i.e.,  a  curve  with  a  dis- 
tinct maximum.  It  is  possible  that  two  processes  are 
involved:  the  general  increasing  reaction  with  rise  of 
temperature,  and  the  destruction  of  the  enzymes  by  heat. 
But  this  is  not  certain.  In  some  enzymes  we  can  see  a 
slow  acceleration  of  action  at  from  io°  to  300  C,  and  a 
much  more  rapid  acceleration  at  from  300  to  370  C.  The 
curve  has  a  form  difficult  to  explain  as  the  summation  of 
opposed  effects.  It  is  possible  that  we  have,  besides  the 
general  acceleration  with  rising  temperature,  a  special 
adaptation  of  the  enzymes  to  the  body-temperature  of 
the  higher  animals.  The  optimum  of  most  of  the  enzymes 
examined  lies  between  300  and  400  C.  But  we  find  old 
statements  that  the  optimum  of  the  diastase  of  yeast  lies  at 
500  C,  or  that  the  pepsin  of  fish  has  an  optimum  between 
20  and  50  C.  These  statements  do  not  hold  good,  as  the 
tissues  of  cold-blooded  animals  contain  proteins  coagulat- 
ing spontaneously  at  300  to  400  C;  if  these  proteins  are 
precipitated,  pepsin  is  precipitated  by  adsorption,  and  the 
extract  becomes  accordingly  poorer  without  change  of  the 
pepsin  itself.  In  the  case  of  yeast,  the  better  solubility 
at  higher  temperatures  can  explain  the  observation. 
Until  we  can  work  with  pure  enzymes,  we  shall  have  great 
difficulties  in  distinguishing  a  change  in  solubility  from  a 
true  change  in  the  enzyme. 

3.  Enzymes  and  Reaction.     Most  enzymes  are  exactly 
adapted  to  the  reaction  of  their  "milieu" — that  is  to  say, 


THE  GENERAL  PROPERTIES  OF  ENZYMES      25 

of  the  solution  in  which  they  occur  in  nature.  Thus 
pepsin  acts  best  in  combination  with  acid,  with  hydrogen- 
ions.  It  does  not  digest  protein,  but  only  the  chloride  of 
the  protein,  and  the  products  of  the  enzyme  are  the 
chlorides  of  peptones.  The  quantity  of  acid  which  is 
bound  on  the  proteins  and  peptones,  and  which  changes 
the  free  compounds  into  chlorides,  is  not  sufficient  for  the 
action  of  pepsin.  This  was  explained  by  Leo.1  Pepsin 
needs  an  excess  of  acid  for  its  action,  that  is  to  say,  it 
needs  free  hydrogen-ions.  When  we  study  dissolved 
protein,  we  cannot  see  this,  because  the  hydrochloric- 
proteins  split  off  acid  by  hydrolysis.  But  if  we  use  a 
solid  protein,  for  instance  fibrin,  we  find  that  we  must  add 
so  much  hydrochloric  acid,  that  the  water,  in  which  the 
swollen  fibrin  lies,  still  contains  acid.  The  natural 
gastric  juice  of  the  dog  or  of  man  contains  one-half  or 
more  per  cent  of  hydrochloric  acid,  but  the  juice  is  diluted 
by  food  to  0.35  per  cent;  it  is  then  1/10  normal  hydro- 
chloric acid,  and  it  has  been  pointed  out  by  Brucke  2 
and  Pawlow,  that  the  optimum  of  pepsin  lies  at  this  lower 
concentration.  The  hydrochloric  acid  can  be  replaced 
by  another  acid  according  to  the  available  hydrogen-ions, 
but  it  seems  that  the  hydrochloric  acid  has  an  exceptional 
value,  which  is  greater  than  that  of  other  acids  of  the 
same  concentration  and  ionization. 

The    necessity    of    co-operation    between    pepsin    and 

1 H.  Leo:     Zeitschr.  f.  physiolog.   Chemie,  46  (1905). — "Die  Salz- 
saure-Therapie,"  Berlin,  1908. 

2E.  Brucke:    Wiener  Akad.,  Math.-Naturw.  Klasse,  37  (1859). 


26  ENZYMES 

hydrochloric  acid  has  caused  many  errors  in  the  chemistry 
of  enzymes.  The  peptones  produced  by  pepsin  are 
basic,  and  neutralize  the  acids;  the  hydrochloric -peptones 
are  hydrolyzed  salts,  and  the  quantity  of  acid  set  free  by 
hydrolysis  depends  upon  the  concentration  and  upon  the 
presence  of  other  neutral  salts.  All  these  influences 
appear  to  us  as  a  diminution  or  an  increase  of  pepsin. 
Furthermore,  we  know  two  proteins,  the  myosin  of 
muscles  or  syntonin,  and  the  fibrin  of  the  blood,  which 
swell  in  acids  and  can  be  attacked  by  pepsin  only  in  the 
swollen  state.  Sodium  chloride  and  -other  neutral  salts 
check  this  swelling,  and  appear  as  opposed  to  pepsin;  as 
"antiferments."  Pepsin  is  least  suitable  as  an  example 
for  pointing  out  the  laws  of  enzymotic  action.  Un- 
fortunately, however,  it  is  used  most  frequently.  The 
relations  of  trypsin  to  the  reaction  have  often  been  in- 
vestigated by  earlier  authors,  but  they  did  not  always 
distinguish  between  the  speed  of  action  of  the  dissolved 
trypsin  and  the  ease  of  dissolving  trypsin  from  the  gland. 
The  enzyme  has  two  actions :  it  dissolves  the  protein,  and 
it  splits  up  proteins  and  peptones  into  simpler  products. 
It  also  seems  to  have  two  different  optima.  It  dissolves 
proteins  when  the  reaction  is  distinctly  alkaline;  and  it 
decomposes  peptones  best  at  the  approximately  neutral  or 
slightly  alkaline  reaction  which  prevails  in  the  intestine. 
This  nearly  neutral  reaction  is  best  also  for  the  enzymes 
of  the  saliva,  the  intestine,  and  the  tissues.  For  a  long 
time  there  has  been  a  division  of  opinion  about  the  re- 
action of  the  contents  of  the  intestine   and  the   tissues. 


THE  GENERAL  PROPERTIES  OF  ENZYMES      27 

The  dispute  arose  solely  because  of  the  deficient  knowledge 
at  the  time  of  the  qualities  of  the  indicators  used  in  the 
study  of  the  reaction.  Now  we  know  that  the  reaction 
of  both  tissues  and  contents  of  the  intestine  resembles  that 
of  a  weak  solution  of  alkali  saturated  with  an  excess  of 
carbonic  acid.  Such  a  solution  gives  the  optimum  for 
the  enzymes  of  the  saliva  and  intestine,  and  for  the 
oxidizing  enzymes  of  the  tissues.  The  tissue  enzymes  are 
destroyed  by  an  abnormal  reaction,  but  the  enzymes  of 
the  intestine  show  a  greater  or  less  resistance  towards 
differences  in  reaction,  a  remarkable  adaptation  to  the 
conditions  of  their  environment,  because  the  reaction  in 
the  upper  duodenum  changes  often  and  suddenly.1 

Zymase,  the  metabolic  enzyme  of  the  yeast,  acts  only, 
or  best,  in  the  presence  of  phosphates;  of  sodium  mono- 
or  diphosphate.  Perhaps  this  is  an  example  of  a  true 
activator,  a  matter  which  will  be  dealt  with  later  on,  but 
more  probably  the  power  of  the  phosphoric  acid  to  hinder 
marked  changes  of  reaction  is  the  cause  of  the  favorable 
influence  of  the  phosphates,  and  this  is  therefore  a  fur- 
ther exemplification  of  the  importance  of  reaction  upon 
ferments. 

4.  Chemical  Properties.  Enzymes  combine  both  with 
their  substrate  and  their  dissociation  products.  The 
specificity  of  the  enzymes  mentioned  before  suggests  to 
us,  that  the  individual  enzymes  must  have  chemical  re- 
lations with  this  substrate.     We  have  no  reason  to  think 

1  H.  Friedenthal:  Zeitschr.  f.  allgem.  Physiologie,  1  (1901). — N.  P. 
Schierbeck:    Skandinav.  Archiv  f.  Physiol.,  3  (1891). 


28  ENZYMES 

that  all  enzymes  belong  to  the  same  class  of  chemical 
compounds.  It  has  already  been  shown  that  the  enzymes 
are  accompanied  as  a  rule  by  proteins  and  nucleic  acids. 
The  difficulties  of  separating  enzymes  and  proteins  have 
led  physiologists  for  a  long  time  to  regard  enzymes  as 
protein-like  bodies,  though  Briicke  *  and  others  2  were 
successful  years  ago  in  freeing  enzymes  from  all  traces  of 
proteins.  Perhaps  steapsin  is  a  fat-like  compound,  and 
sucroclastic  enzymes  sugar-like  compounds,  and  perhaps 
they  belong  to  a  wholly  different  class.  We  know  but 
little  to-day  about  the  chemical  characteristics  of 
enzymes. 

Enzymes  are  salted  out  by  such  neutral  salts  as  are 
capable  of  readily  salting  out  proteins,  uric  acid,  and 
many  dye-compounds,  and  particularly  by  ammonium 
sulphate.  It  seems  that  the  individual  enzymes  are 
precipitated  at  different  concentrations  of  ammonium 
sulphate — aldehydase  and  erepsin,  when  the  solution 
contains  sixty  per  cent,  and  trypsin  and  invertin  at 
complete  saturation.  These  differences  can  be  used 
practically  for  purifying  and  separating  enzymes,  but 
they  throw  no  light  on  their  chemical  nature.  Enzymes 
are  precipitated  by  alcohol,  and  the  individual  enzymes 
apparently  by  different  concentrations  of  alcohol.  En- 
zymes are  precipitated,  further,  by  heavy  metals  like 
uranium,  and  they  are  precipitated  by  tannin  and  by 

1  E.  Briicke:    Wiener  Akad.,  Math.-Naturw.  Klasse,  1861. 

2  J.  Cohnheim:  Virchow's  Archiv,  28  (1863). — M.  Jacoby;  Zeitschr. 
f.  physiol.  Chemie,  30  (1900). 


THE  GENERAL  PROPERTIES  OF  ENZYMES      29 

phosphotungstic  acid.  The  enzymes  can  be  dissolved 
again  immediately  after  precipitation,  but  on  standing 
they  readily  lose  their  solubility.  Color  tests  for  enzymes 
are  not  known. 

It  is  of  practical  importance  that  enzymes  are  neither 
precipitated  nor  destroyed  by  many  antiseptic  substances, 
i.e.,  by  substances  that  kill  and  destroy  living  cells  or 
check  their  growth.  Chloroform,  toluene,  thymol,  and 
ether,  which  dissolve  the  lipoid  substances  in  cells  and 
bacteria,  and  therefore  destroy  structures  necessary  to 
life,  exert  no  influence  on  enzymes,  because  the  enzymes 
are  completely  soluble  in  water,  and  do  not  yield  any 
lipoids.  It  is  very  difficult  to  get  extracts  of  living  tissues, 
which  cannot  be  heated  or  boiled,  free  from  bacteria;  so 
far  as  concerns  the  liver  and  pancreas,  which  yield  bacteria 
in  life,  it  is  impossible. 

During  life,  growth  of  micro-organisms  is  prevented  or 
controlled  in  organs,  but  after  death  and  loss  of  structure, 
bacteria  grow  in  the  fluids  rich  in  proteins,  carbohydrates, 
and  suitable  salts.  These  bacteria  split  the  proteins, 
carbohydrates,  and  fats,  as  do  the  hydrolytic  ferments, 
and  give  rise,  like  zymase,  to  carbon  dioxide.  Every  one 
who  has  worked  with  ferments  knows  how  easily  we  can 
be  deceived  when  led  to  suppose  ferments  to  be  present 
when  in  fact  we  are  dealing  with  bacteria.  We  distin- 
guish between  the  two  when  we  allow  ferments  to  work 
for  only  two  or  three  hours,  because  the  action  of  ferments 
proceeds  very  quickly,  whilst  the  growth  of  bacteria,  even 
under  the  most  favorable  conditions,  takes  much  more  time. 


30  ENZYMES 

But  during  extraction  and  purification,  and  if  we  extend 
experiments  for  a  long  time,  it  is  absolutely  necessary  to 
protect  enzyme  solutions  against  bacteria  by  means  of 
antiseptic  substances.  Amongst  these  chloroform  or 
toluene  have  been  most  used  within  the  last  few  years,  and 
for  the  study  of  the  enzymes  of  the  alimentary  canal 
crowded  with  bacteria,  I  suggest  using  both  substances 
simultaneously.  Thymol  and  ether  have  no  efficiency. 
I  think  that  all  observations  on  enzymes,  made  without 
any  addition  of  antiseptics,  must  be  regarded  with  doubt 
and  distrust.  Other  antiseptics,  which  coagulate  proteins, 
such  as  fluorides,  potassium  meta-arsenite,  and  mercuric 
chloride,  seem  to  have  an  injurious  effect  on  the  action  of 
the  enzymes.  But  no  evidence  has  been  brought  forward 
showing  whether  ferments  are  precipitated  and  destroyed 
by  these  compounds,  or  whether  the  proteins,  on  coagulat- 
ing, absorb  and  withdraw  the  enzymes.  Buchner  *  has 
observed  that  zymase  does  not  lose  power  in  the  presence 
of  potassium  meta-arsenite  or  ammonium  fluoride  in  small 
amount,  and  that  the  proteolytic  ferments  of  leucocytes 
are  not  harmed  by  corrosive  sublimate.  But  these  solu- 
tions, zymase  and  the  extracts  of  leucocytes,  are  rich  in 
protein,  and  the  harmful  influence  of  the  mercury  or  the 
arsenite  is  checked  by  their  union  with  protein.  Purified 
enzyme  solutions  have  not  yet  been  studied  with  regard 
to  their  sensitiveness  to  these  antiseptics,  but  for  most 
practical  purposes  the  lipoid  antiseptics  are  thoroughly 
satisfactory. 

1  E.  H.  Buchner:     "Zymasegarung,"  Munich,  1903. 


CHAPTER   IV 


Enzymes  as  Catalyzers 


We  know  of  chemical  processes  in  which  the  reacting 
substances  completely  disappear,  giving  rise  to  a  new 
body.  But  in  other  processes  one  chemical  compound 
reacts  with  another  compound  and  changes  it  without 
entering  into  the  final  reaction  itself.  For  instance,  the 
conversion  of  cane  sugar  is  expressed  by  the  equation: 

C12H22On+H20  +  wHCl  =  C6H1206  +  C6H1206  +  »HC1. 

The  hydrochloric  acid  causes  the  reaction,  but  before 
and  after  the  reaction  we  find  the  same  quantity  of  acid. 
A  second  case  is  the  formation  of  ether  from  alcohol  by 
sulphuric  acid : 

2C2H5OH  +  wS04H2  =  C2H5.O.C2H5  +  H20  +  «S04H2. 

The  sulphuric  acid  is  the  cause  of  the  reaction,  but 
neither  the  original  alcohol  nor  the  end-products  of  the 
reaction  contain  the  sulphuric  acid,  and  the  most  import- 
ant point  is  the  fact  that  there  is  no  quantitative  relation 
between  the  hydrochloric  acid  and  the  sugar  or  between 
the  sulphuric  acid  and  the  alcohol  and  ether.  Chemists  * 
apply  the  term  "catalytic"  to  such  reactions,  and  it  seems 
that  all  enzymotic  processes  are  catalytic  reactions.     The 

1  G.  Bredig:    Ergebnisse  der  Physiologie,  i,  Biochemie  (1902). 

31 


32  ENZYMES 

evidence  for  their  designation  as  catalyzers  is  that  we 
cannot  find  any  relation  between  the  quantity  of  enzyme 
and  the  quantity  of  matter  decomposed  by  it,  and  that, 
after  the  reaction,  we  find  the  enzyme  is  not  consumed. 
The  last  argument,  however,  is  not  completely  proved, 
because  the  estimation  of  the  enzymes  is  not  so  exact  as 
to  exclude  the  consumption  of  a  portion. 

The  great  discrepancy  between  the  small  quantity  of 
enzymos  and  the  enormous  quantity  of  matter  converted 
by  enzymes,  is  astonishing,  and  has  impressed  all  in- 
vestigators. The  best  explanation  seems  to  be  the  ad- 
mission of  an  intercalated  reaction  assumed  by  chemists 
generally  in  the  case  of  the  formation  of  ether.  Thus  the 
reaction  would  be  expressed  by  two  equations : 

i.  C2H5OH  +  S04H2  =  C2H5  (HSOJ  +  H20. 

2.  C2H5  (HS04)  +  C2H5OH  =  C2H5 . 0 .  C2H5  +  H2S04. 

That  is  to  say,  the  molecule  of  the  sulphuric  acid  is 
combined  first  with  one  molecule  of  alcohol,  then  it 
combines  with  a  second  molecule  of  alcohol,  and  finally 
it  is  liberated  and  can  attack  a  new  molecule  of  alcohol. 
It  is  highly  probable  that  enzymes  are  combined  thus  with 
the  substrate  they  work  upon  and  with  the  products  they 
yield.  The  arguments  for  this  supposition  will  be  cited 
later  on,  but  since  I  have  spoken  of  the  enzymes  as  cataly- 
zers, the  theory  of  Ostwald  and  Bredig  should  also  be 
mentioned.  They  have  ascribed  a  new  function  to  cata- 
lyzers besides  the  quality  mentioned.  They  define  a 
catalyzer  as  a  substance  that  never  causes  a  reaction,  but 


ENZYMES   AS   CATALYZERS  33 

only  accelerates  a  reaction  going  on  spontaneously  but 
more  slowly  without  the  catalyzer.  They  propose  the 
theory  that  the  proteins  or  carbohydrates  are  always 
slowly  dissociated  in  watery  solution,  and  that  the  enzymes 
hasten  this  dissociation.  They  thought  that  through  this 
theory  they  could  avoid  the  difficulty  presented  by  the 
fact  that  enzymes  mediate  in  such  extensive  processes, 
extensive  also  as  regards  the  energy  involved.  They 
argue  that  our  body  produces  its  whole  energy  through 
the  enzymes  oxidizing  and  burning  the  food,  and  that  it 
is  difficult  to  suppose  that  the  great  quantity  of  energy — 
four  large  calories  per  gramme  of  glucose,  and  nine  per 
gramme  of  fat — can  be  liberated  by  the  small  quantity 
of  enzymes  which  are  available.  The  argument  of 
Ostwald  and  Bredig  has  been  adopted  by  many  investi- 
gators; in  fact  we  may  say  that  it  has  ruled  the  whole 
theory  of  enzymotic  action  in  recent  years,  but  it  seems  to 
me  that  it  does  not  hold  good,  and  for  three  reasons: 

i.  It  is  not  true  that  proteins  or  carbohydrates  spon- 
taneously undergo  a  slow  dissociation  in  solution.  Such 
solutions  have  been  preserved  for  seventeen  years  or  more 
at  room  temperature,  and,  in  the  absence  of  micro-organ- 
isms, of  acid,  or  of  alkali,  the  substances  have  been  found 
unchanged.  We  have  no  reasons  for  thinking  that  the 
time  of  observation  is  too  short,  and  that  we  would  find 
a  dissociation  in  centuries. 

2.  If  we  heat  cane  sugar  with  acid,  it  is  decomposed  in 
the   same   manner  as  by  invertin,   and  we   could   here 
attribute  the  decomposition  really  to  the  H-ions,  which 
3 


34  ENZYMES 

neutral  water  contains  in  lowest  concentration,  and  assume 
simply  that  the  enzyme  hastens  it.  But  in  other  cases, 
the  enzymes  cause  other  reactions  than  the  H-ions  of  the 
water  would  bring  about.  The  dissociation  of  glucose 
produced  by  H-  or  OH-ions  gives  rise  to  lactic  acid  and 
humins,  while  the  dissociation  by  zymase  produces 
alcohol. 

3.  Sugars  and  fats  are  oxidized  and  burned  in  the 
tissue,  and  set  free  much  energy,  but  we  do  not  know 
exactly  to  what  extent  this  oxidation  is  caused  by  enzymes, 
and  especially  by  one  enzyme.  It  is  possible  that  the 
structure  of  cells  is  needed  for  the  complete  combustion, 
and  that  two,  three,  or  more  enzymes  work  consecutively 
and  perhaps  in  separate  localities.  We  know  that  zymase 
is  the  only  enzyme  that  gives  off  heat,  and  only  a  very 
small  quantity  of  heat.  The  well:known  enzymes,  the 
hydrolytic  ones,  liberate  no  heat.  The  action  of  trypsin 
was  measured  by  Grafe  *  in  Rubner's  laboratory  with  a 
calorimeter  of  high  sensitiveness.  He  found  no  rise  of 
temperature.  The  other  enzymes  have  not  been  measured 
in  this  manner,  so  far  as  I  know,  but  the  heat  of  combustion 
of  starch,  the  disaccharides,  and  of  glucose,  has  been 
determined,  and  we  can  calculate  that  the  differences  lie 
within  the  errors  of  analysis.  The  building-up  of  protein 
from  the  amino-acids,  or  of  the  polysaccharides  from  the 
hexoses,  as  well  as  the  accompanying  dissociations,  seems 
to  be  unconnected  with  any  perceptible  thermic  process. 


1  E.  Grafe:    Archiv  f.  Hygiene,  62  (1907). 


ENZYMES   AS   CATALYZERS  35 

It  is  of  great  importance  for  the  understanding  of  enzymes, 
that  the  hydrolytic  enzymes  neither  give  out  nor  consume 
any  considerable  or  perceptible  quantities  of  heat.  Opin- 
ions regarding  energy  have  here  no  weight. 

We  must  see  whether  well-observed  facts  support  the 
theory  that  enzymes  are  like  inorganic  catalyzers,  and 
only  hasten  reactions  without  provoking  them.  For  all 
proteolytic  enzymes  no  fact  is  known  which  gives  any 
evidence  for  it,  and  the  same  is  true  of  ptyalin,  invertin, 
lactase,  zymase,  and  the  corresponding  metabolism  en- 
zymes urease  and  nuclease.  On  the  other  hand,  evidence 
has  been  brought  forward  that  esters  like  fats  are  dissociat- 
ed spontaneously  by  the  ions  of  the  water,  and  that  steap- 
sins  or  lipases  which  split  up  fats  and  other  esters,  only 
hasten  this  process.  Later  on,  in  treating  of  the  synthetical 
action  of  enzymes,  I  shall  discuss  the  steapsins,  or  lipases, 
which  are  distinguished  from  all  other  enzymes,  because 
they  alone  effect  synthesis  to  any  great  extent.  I  think 
that  the  capacity  for  acting  synthetically  is  connected  with 
the  property  of  the  steapsins  to  hasten  only  a  slow  spon- 
taneous action.  The  steapsin  seems  to  have  the  proper- 
ties of  catalyzers  in  the  sense  of  Ostwald  and  Bredig,  and 
this  property  lies  at  the  bottom  of  the  synthetical  action. 
According  to  the  most  important  work  of  Pottevin,1  the 
relations  between  dissociations  and  synthetical  action  in 
steapsin  are  perfectly  similar  to  the  relations  between 
alcohols,   acids,    and   esters   in   watery   solution  without 

1  Pottevin:    Annales  de  PInstitut  Pasteur,  20  (1906). 


36  ENZYMES 

enzymes,  because  both  reactions  show  an  equilibrium- 
point,  which  is  moved  in  the  one  or  the  other  direction 
according  to  the  temperature  or  the  presence  of  more  or 
less  water.  If  ethyl  alcohol  is  heated  with  acetic  acid, 
the  following  reaction  occurs : 

CH3COOH  +  C2H5OH  =  CH3CO.O.C2H5  +  H20. 

But  if  water  and  ethyl  acetate  are  mixed,  the  opposite 
reaction  takes  place,  as  follows : 

CH3CO.O.C2H5  +  H20  =  CH3COOH  +  C2H5OH. 

If  these  two  substances,  alcohol  and  acetic  acid,  are 
allowed  to  mix,  both  reactions  must  proceed  simultaneously 
in  the  same  solution  until  they  reach  an  equilibrium-point, 
that  is  to  say,  the  point  at  which  both  proceed  at  the  same 
rate,  and  all  change  seems  to  be  stopped.  The  position 
of  the  equilibrium-point  depends  upon  the  mass  of  the 
three  reacting  substances,  water,  acid,  and  alcohol,  and 
it  depends,  further,  upon  the  temperature  of  the  solution. 
The  equation  is  therefore  expressed  in  this  manner: 

CH3COOH  +  C2H5OH^CH3CO.O.C2H5  +  H20. 

The  steapsin  moves  the  equilibrium-point,  and  thus 
acts  like  a  true  catalyzer.  In  concentrated  solutions  it 
hastens  synthesis,  but  if  we  add  water,  it  hastens  disso- 
ciation. It  is  remarkable  now,  that  the  disaccharides, 
maltose  and  isomaltose,  are  glucosides  according  to  E. 
Fischer,  and  are  similar  to  ether  in  structure,  and  that  he 
even  has  observed  strong  hydrochloric  acid  to  have  a 
synthetical  influence  on  glucose.     Compounds  like  isomaL 


ENZYMES   AS   CATALYZERS  37 

tose  are  thus  formed.  No  evidence  has  been  brought 
forward  thus  far  regarding  the  slow  dissociation  of  maltose 
in  the  absence  of  maltase,  but  perhaps  we  can  expect  this 
fact  to  be  observed,  and  in  that  case  maltase  must  be 
likened  to  steapsin  and  not  to  sucroclastic  enzymes  which 
attack  the  anhydrous  polysaccharides  like  starch  or  cane 
sugar,  and  which  exhibit  no  catalytic  quality. 

A  second  class  of  enzymes,  which  hasten  only  those 
reactions  proceeding  slowly  without  them,  are  the  oxidases 
of  the  type  of  laccase.  The  more  important  metabolic 
enzymes,  like  zymase  or  lactacidase,  I  repeat  emphatically, 
provoke  conversions  which  do  not  occur  at  all  in  the 
absence  of  enzymes,  and  so  do  all  proteolytic  enzymes  and 
the  nucleases. 

Bredig,  Henri,  and  others  have  compared  the  time 
velocity  of  reactions,  which  are  produced  by  inorganic 
catalyzers  and  by  enzymes,  and  they  have  found  that  the 
curves  resemble  one  another,  but  show  some  differences. 
I  have  already  stated  that  it  is  impossible  to  study  the 
laws  of  action,  if  we  have  only  solutions  so  crowded  with 
impurities  as  are  our  enzyme  solutions.  I  have  said,  and 
may  repeat  again,  that  enzymes  are  modified  in  all  sorts 
of  ways  in  solutions,  and  shall  presently  mention  that  the 
action  of  enzymes  is  checked  by  the  products  formed  by 
their  action,  and  occurring  therefore  in  the  solutions; 
while  in  the  case  of  ether  or  cane  sugar,  the  acids  pro- 
voking reactions  are  not  influenced  by  substances  appear- 
ing in  the  solution.  Thus  irregular  curves  result,  which 
throw  light  upon  the  happenings  in  enzymotic  processes. 


38  ENZYMES 

We  can  only  observe  that  the  action  of  enzymes  begins 
immediately  when  they  touch  their  substrate,  and  that 
the  process,  with  suitable  reaction  and  temperature, 
proceeds  most  rapidly.  If  we  add  saliva,  containing 
ptyalin,  to  starch,  reducing  sugars  can  be  detected  as  soon 
as  the  fluids  are  mixed.  Fibrin  is  dissolved  in  gastric 
juice,  containing  pepsin,  as  rapidly  as  sugar  dissolves  in 
water.  If  we  allow  active  trypsin  to  act  upon  casein  or 
other  easily  digestible  protein,  tyrosine  is  split  off  in  so 
short  a  time  that  it  resembles  a  mere  precipitation  or 
crystallization  of  the  insoluble  amino-acid.  When  study- 
ing arginase,  Kossel  wished  to  heat  a  liver  extract  with 
an  enzyme  in  order  to  destroy  arginase,  and  he  set  the 
tube  in  boiling  water.  The  short  time  before  the  extract 
became  heated  was  sufficient  for  the  arginase  to  act  upon 
the  arginine  and  to  split  off  some  urea.  The  glycolytic 
enzyme  of  muscles  does  not  convert  more  sugar  in  twelve 
hours  than  in  two.  The  quick  beginning  and  the  rapid 
progress  is  a  special  feature  of  enzymotic  action.  If  I 
find  a  ferment  that  acts  only  after  a  long  time,  proceeds 
slowly,  and  forms  end-products  only  in  small  quantity,  I 
always  conclude  that  either  the  enzyme  is  of  no  great 
importance,  or  is  not  present  under  suitable  conditions. 


CHAPTER  V 

The  Reversible  Action  of  Enzymes 

We  know  of  many  synthetic  processes  in  the  animal 
body  which  are  opposed  to  the  hydrolytic  processes  caused 
by  enzymes.  We  can  extract  from  the  liver  a  diastase  and 
a  maltase  dissociating  glycogen  into  maltose,  and  maltose 
into  glucose;  the  normal  active  liver  can  again  convert 
glucose  into  glycogen.  We  can  extract  from  the  kidney 
the  histozyme  which  splits  hippuric  acid  into  glycocoll  and 
benzoic  acid,  while  the  living  or  surviving  kidney  builds 
up  hippuric  acid  from  the  cleavage  products.1  The 
mucous  membrane  of  the  small  intestine  secretes  a 
steapsin  which  decomposes  fats;  and  the  mucous  mem- 
brane builds  up  neutral  fat  from  the  fatty  acid  and  the 
glycerin  entering  the  cells.  Proteins  are  formed  in  the 
body  from  the  amino-acids  or  from  material  still  further 
removed  from  the  protein.  Plants  decompose  and  build 
up  cane  sugar  and  starch  according  to  their  needs,  and 
lecithin  and  nucleic  acid  arise  in  the  developing  egg 
from  substances  which  are  quite  different.2  For  a  long 
time  it  was  thought  that  perhaps  enzymes  could  also  cause 
these  opposite  synthetic  reactions.     For  the  theory  which 

1  G.  Bunge  and  O.  Schmiedeberg:  Archiv  f.  exper.  Path.  u.  Pharm., 
6  (1876). 

2  A.  Kossel:    Zeitschr.  f.  physiolog.  Chemie,  10  (1886). 

39 


40  ENZYMES 

regards  enzymes  as  catalyzers,  and  enzyme-reactions  as 
equilibrium-reactions,  it  seems  necessary  to  assume  the 
synthetic  action  of  enzymes. 

The  first  to  report  an  experimental  proof  of  a  reversible 
synthetical  action  of  an  enzyme  was  Croft  Hill,1  in  1898. 
He  observed  that  in  a  concentrated  solution  of  glucose 
(forty  per  cent),  treated  with  a  water-extract  of  yeast 
containing  maltase,  the  rotatory  and  reducing  power 
changes  after  a  few  weeks.  The  fact  has  been  supported 
by  Emmerling,2  and  it  seems  to  be  certain  that  extracts 
of  yeast  and  plants  containing  maltase  convert  glucose  in 
highly  concentrated  solutions  to  a  greater  or  less  amount, 
and  give  rise  to  a  disaccharide  and  to  dextrin-like  bodies. 

The  nature  of  this  synthetic  disaccharide  is  debated;  it 
seems  that  it  is  not  the  maltose  hydrolyzed  by  maltase, 
but  an  isomeric  substance,  isomaltose,  occurring  also 
during  the  hydrolysis  of  starch  and  glycogen,  but  not 
hydrolyzed  by  maltase.  We  would  have  here,  therefore, 
a  synthetic  action,  but  not  a  reversible  action  of  the  maltase. 
Strong  hydrochloric  acid  seems  to  act  in  the  same  manner 
as  the  synthetizing  enzyme.  E.  Fischer  and  Armstrong  3 
made  a  similar  observation  on  treating  concentrated 
solutions  of  glucose  with  lactase,  i.e.t  with  an  extract  of 
kefir  grains.     They  found  a  disaccharide,  not  the  lactose, 


1  A.  Croft  Hill:    Transactions  of  the  Chem.  Soc,  73  (1898).— Ber.  d. 
deutsch.  chem.  Ges.,  34  (1901). 

2  O.  Emmerling:    Ber.  d.  deutsch.  chem.  Ges.,  34  (1901). 

3  E.  Fischer  and  E.  F.  Armstrong:     Ber.  d.  deutsch.  chem.  Ges.,  35 
(1902). 


THE   REVERSIBLE   ACTION   OF   ENZYMES  41 

milk  sugar,  but  an  isomeric  compound  resembling  it, 
which  they  named  isolactose.  This  isolactose  is  not 
hydrolyzed  by  the  lactase,  which  builds  it  up.  According 
to  Buchner,1  a  great  amount  of  the  glucose  added  to  the 
press-juice  of  yeast  is  dissociated  into  alcohol  and  carbon 
dioxide,  but  another  portion  is  converted  into  dextrin-like 
bodies.  Cremer  2  observed  that  in  the  cell-free  press-juice 
glycogen  is  formed  from  glucose. 

We  are  much  better  informed  regarding  the  synthetic 
action  of  the  fat-splitting  enzymes.  Kastle  and  Loeven- 
hart 3  were  the  first  to  observe  that  in  a  mixture  of  butyric 
acid  and  ethyl  alcohol,  ethyl  butyrate  is  formed  when  a 
fresh  pancreas  extract  containing  steapsin  is  added.  A 
control  experiment  carried  out  with  boiled  pancreas  ex- 
tract gave  no  ester  whatever  because  the  ethyl  butyrate, 
like  other  ethers,  is  hydrolyzed  by  the  steapsin  of  the 
pancreas.  Here  the  synthesis  and  the  hydrolysis  involve 
the  same  compounds.  The  observation  of  Kastle  and 
Loevenhart  was  confirmed  by  several  observers,  and  in 
1906  Pottevin,4  in  Paris,  succeeded  in  obtaining  a  synthesis 
of  the  true  fats  in  large  amount  by  means  of  the  steapsin 
or  the  lipase  of  pancreas.  He  treated  finely  divided 
pancreas  with  alcohol  and  ether,  and  thus  prepared  a 
fine  dry  powder  which  was  insoluble  in  water,  but  which 
contained  the  lipase.     Then  he  made  a  mixture  of  100 

1  E.  and  H.  Buchner  and  M.  Hahn:"  Die  Zymasegarung,"  Munich,  1903. 

2  M.  Cremer:    Ber.  d.  deutsch.  chem.  Ges.,  32  (1899). 

3  J.  H.  Kastle  and  A.  Loevenhart:    Amer.  Chem.  Journ.,  24  (1900). 

4  Pottevin:    Annal.  de  l'lnstitut  Pasteur,  20  (1906). 


42  ENZYMES 

gm.  pure  oleic  acid,  a  little  water,  and  the  calculated 
quantity  of  glycerin,  and  added  2.5  gm.  of  the  pancreas 
powder.  The  mixture  was  kept  from  three  to  twenty 
days  at  330  C.  In  that  time  85  gm.  of  the  oleic  acid  were 
converted  into  mono-olein;  in  another  series,  more  than 
10  gm.  of  triolein  were  formed.  Controls  demonstrated 
that  without  the  pancreas-powder,  or  with  boiled  pancreas- 
powder,  only  a  small  amount  of  the  ester  (four  per  cent 
as  against  eighty-eight  per  cent)  was  formed,  and  that  the 
velocity  of  the  reaction  depends  upon  the  quantity  of  the 
enzyme  powder.  If  more  water  is  added,  the  quantity 
of  the  ester  formed  diminishes,  and  in  dilute  solution  the 
mono-olein  and  triolein  are  hydrolyzed  by  the  same  lipase. 
The  reaction  induced  by  the  lipolytic  enzyme  is  a  strictly 
reversible  one. 

The  proteins,  peptones,  and  peptids  are  not  built  up  by 
enzymes,  nor,  so  far  as  we  know,  are  urea,  hippuric  acid, 
or  cane  sugar.  It  seems  that  the  reversible  action  occurs 
only  with  esters,  that  is  to  say,  with  compounds  which, 
themselves,  and  without  any  enzyme,  show  a  typical 
reversibility,  a  typical  equilibrium-reaction.  The  equili- 
brium-point in  dilute  solutions  approaches  the  limit  of 
dissociation,  while  in  concentrated  solutions  it  approaches 
the  other  side  of  the  equation.  The  enzymes  accelerate 
the  reaction  or  move  the  point  of  equilibrium  farther  in 
one  direction  than  it  would  lie  without  them.  The  fats 
are  esters,  and  it  was  shown  by  Nencki l  that  lipases  or 

1  M.  Nencki:    Archiv  f.  exper.  Pathol,  u.  Phar.,  20  (1885). 


THE   REVERSIBLE   ACTION   OF   ENZYMES  43 

steapsins  also  decompose  many  other  esters.  Kastle 
and  Loevenhart,  Pottevin,  and  others  have  observed  that 
they  also  synthetize  other  esters  from  dissociation-pro- 
ducts. The  disaccharides,  or  maltose,  isomaltose,  lactose, 
and  isolactose,  are,  according  to  E.  Fischer,  glucosides,  or 
aldehyde-sugars,  and  their  combinations  are  ethers, 
resembling  the  esters  in  behavior.  Cane  sugar  and  the 
higher  polysaccharides  are  anhydrous  sugars,  and  not 
ethers,  and  with  them  no  synthesis  has  been  observed. 

We  must  very  carefully  separate  enzymes  into  two 
classes.  The  one  class,  the  catalytic,  exhibits  synthetic 
power,  and  it  is  possible  that  the  animal  body  uses  them 
for  building  up  fats  and  other  compounds.  With  the 
other  class,  not  the  slightest  trace  of  synthesis  has  yet 
been  observed. 


CHAPTER  VI 

Enzymes  and  Optical  Activity 

Pasteur  was  the  first  to  observe  that  the  two  optically 
active  forms  of  a  chemical  compound  can  differ  in  their 
behavior  during  metabolism.  He  noticed  that  micro- 
organisms, e.g. }  Penicillium  glaucum,  attack  only  one  of 
the  two  isomers  of  tartaric  acid,  and  leave  the  other  un- 
attacked.  Later,  E.  Fischer1  discovered  the  constitution 
of  sugars;  he  was  able  to  buildup  synthetically  all  known 
hexoses,  and  many  others  not  occurring  naturally,  and 
he  threw  light  upon  the  constitution  of  the  disaccharides. 
After  this,  he  studied  the  action  of  the  hydrolyzing  enzymes 
of  yeast  and  the  higher  animals,  and  the  enzymes  of  yeast 
causing  fermentation.  The  sixteen  possible  stereo- 
isomeric  hexoses  differ  only  by  the  position  of  the  H  and 
OH  in  space,  and  Fischer  found  that  of  the  common 
hexoses  only  four  are  fermented  by  yeast.  I  here  give 
the  graphic  formulae  of  these  four  sugars  (see  facing 
page). 

Glucose,  mannose,  and  levulose  do  not  differ  in  their 
four  last  carbon  atoms.  Galactose  presents  a  slight  dif- 
ference in  the  fourth  atom,  and  we  find  it  is  fermented 

1  E.  Fischer:  Ber.  d.  deutsch.  chem.  Ges.,  23  (1890);  27  (1894).  The 
enzymes  are  treated  of  in  vol.  27  and  28,  and  Zeitschr.  f.  physiol.  Chem., 
26  (1898). 

44 


ENZYMES   AND    OPTICAL  ACTIVITY 


45 


COH  COH 

I  I 

H-C— OH  HOC— H 

I  I 

HOC— H  HOC— H 


CH2OH  COH 

I  I 

C  =  0  H— C— OH 

HOC— H  HO— C— H 


H— C— OH     H— C— OH    H— C— OH    HO— C— H 
H— C— OH     H— C— OH    H— C— OH      H— C— OH 


CH2OH 

Glucose 


CH2OH 
Mannose 


CH2OH 
Levulose 


CH2OH 

Galactose 


more  slowly  than  the  other  three.  All  the  other  known 
possible  hexoses  are  left  unaffected  by  yeast.  Still 
clearer  are  the  relations  between  optical  activity  and  the 
hydrolyzing  enzymes.  There  are  three  enzymes  which 
dissociate  disaccharides — invertin,  maltase,  and  lactase. 
According  to  E.  Fischer,  in  the  disaccharides  hexoses  are 
linked  as  in  the  glucosides,  and  he  has  built  up  from  glu- 
cose  and  methyl  alcohol  two  artificial  stereo-isomeric  glu- 
cosides resembling  the  natural  glucosides,  the  a-  and 
/3-methylglucosides. 


H.C.OCHc 


H.C.OH 

I        N 
HO.C.H  / 

I    / 
H.C/ 

I 
H.C.OH 


O 


CH3O.C.H 

l\ 
H.C.OH 

HO.C.H   /° 

I    / 
H.C/ 

I 
H.C.OH 


CH2OH  CH2OH 

a-methylglucoside      /3-methylglucoside 


46  ENZYMES 

The  two  glucosides  are  identical  except  in  the  spatial  or 
steric  position  of  the  radicals  in  the  first  carbon  atom. 
Of  these  two  compounds,  invertin  splits  the  a-glucoside 
and  lactase  and  emulsin  the  /?-glucoside.  Both  leave 
untouched  the  compound  optically  opposite.  E.  Fischer 
has  also  built  up  the  two  active  ethyl-glucosides  and  they 
are  decomposed,  the  a-glucoside  by  the  one,  and  the  <5- 
compound  by  the  other,  enzyme.  The  glucosides  occurring 
naturally  in  plants  are  /?-glucosides;  they  are  all  decom- 
posed only  by  emulsin  or  by  lactase,  and  not  by  invertin. 

E.  Fischer  1  has  also  built  up,  by  linking  amino-acids, 
chains  which  he  calls  "  peptids."  The  amino-acids  arising 
from  proteins  are  optically  active.  For  the  synthesis 
Fischer  took  both  the  amino-acids  occurring  in  nature, 
and  the  optically  opposite  forms,  and  also  inactive  racemic 
compounds.  In  studying  the  action  of  proteolytic 
enzymes,  trypsin  and  erepsin,  on  these  bodies,  he  found 
that  they  attacked  only  those  compounds  which  corre- 
spond to  the  substances  occurring  in  nature.  The 
optically  opposite  form  is  not  affected,  and  only  half  of 
the  racemic  form  is  decomposed.  These  relations  are 
analogous  to  those  pertaining  to  carbohydrates. 

The  large  protein  molecule  exhibits  two  ways  of  linking 
amino-acids,  the  amino-linking  which  occurs  in  the 
peptids,  and  the  amino-linking  which  unites  urea  and 
ornithine  in  arginine.     The  arginine  has  its  own  enzyme, 

1  E.  Fischer:  Ber.  d.  deutsch.  chem.  Ges.,  33-42,  and  Fischer:  "  Un- 
tersuchungen  iiber  Aminosauren,  Polypeptide  und  Prote'ine,"  Berlin, 
too6. 


ENZYMES   AND   OPTICAL  ACTIVITY  47 

arginase,  discovered  by  Kossel  and  Dakin.1  The  natural 
arginine  and  one  of  its  cleavage-products,  ornithine,  are 
dextro-rotatory.  The  arginase  decomposes  only  this 
dextro-rotatory  arginine.  If  we  take  racemic  arginine, 
only  half  is  split,  and  Kossel  and  Riesser 2  were  thus  able 
to  isolate  the  levorotatory  arginine  until  then  unknown. 

The  efficiency  of  enzymes,  at  least  of  the  enzymes  that 
attack  optically  active  substances,  depends  upon  the 
steric  configuration  of  the  molecules  they  attack.  But 
the  steric  configuration  is  not  the  only  restriction  by  which 
they  are  bound.  The  a-methylglucoside,  which  I  have 
mentioned,  contains  glucose,  a  hexose;  and  Fischer  has 
built  up  in  a  similar  manner  the  a-methylxyloside,  in 
which  methyl  alcohol  and  xylose,  a  pentose,  are  linked 
together.  I  give  here  the  formulae  of  the  glucoside  and 
of  the  xyloside.  We  see  that  the  xyloside  differs  only 
by  the  absence  of  the  sixth,  or  perhaps  better  the  fifth, 
C-atom. 

H.C.OCH3  H.C.OCH3 

l\  l\ 


H.C.OH 

H 

/ 


HO.C.H  )°  HO.C.H  /° 


H.C  H.C 

I  I 

H.C.OH  CH2OH 

I 
CH2OH 

Glucoside  Xyloside 


1  A.  Kossel  and  H.  D.  Dakin:  Zeitschr.  f.  physiol.  Chem.,  41,  42  (1904). 

2  O.  Riesser:     Ibid.,  49  (1906). 


. 


48  ENZYMES 

Invertin  loosens  the  link  between  the  glucose  and  the 
methyl  alcohol,  that  is  to  say,  it  attacks  the  molecules  at 
a  place  where  no  difference  exists  between  the  glucoside 
and  the  xyloside,  nevertheless  the  invertin  does  not  dis- 
integrate the  xyloside. 

Another  instance:  In  the  organism  of  the  higher 
animals,  we  know  of  three  combinations  between  the 
COOH-group  and  the  NH2-group,  with  loss  of  water. 
In  the  three  cases  the  two  groups  are  themselves  linked 
together  in  the  same  manner,  but  the  substitutions  in  these 
groups  differ.  Here  are  the  formulae  for  urea,  glycyl- 
glycine,  the  simplest  peptid,  and  hippuric  acid. 

O 

II     H 
H2N— C-fNH  =  Urea 

O  O 

H     ||     H    H     || 
H2N— C— C-hN— C— C— OH  =  Glycyl-glycine 
H  H 

O  O 

II  .  H    H     || 
H5C—  C4-N— C— C— OH  =  Hippuric  Acid 
H 

The  three  compounds  are  split  at  the  place  I  have  indicated 
and  we  can  see  no  difference  in  the  molecules  directly  next 
the  line  of  splitting.  In  the  case  of  urea,  we  have  merely 
the  NH2-group;  in  the  other  compounds  one  H  is  sub- 
stituted, the  right  side  being  the  same  in  the  glycyl-glycine 
and  the  hippuric  acid.     Nevertheless,  every  one  of  the 


ENZYMES   AND   OPTICAL  ACTIVITY  49 

three  compounds  has  its  own  individual  enzyme.  The 
urea  is  decomposed  by  certain  bacteria  containing  urease. 
The  hippuric  acid  is  dissociated  by  histozyme,  an  enzyme 
found  in  the  kidney  by  Schmiedeberg.1  The  glycyl-glycine 
is  attacked  by  erepsin,  an  enzyme  produced  by  the  small 
intestine  and  other  tissues.  The  histozyme  cannot  affect 
the  peptids,  nor  can  the  erepsin  attack  hippuric  acid.2 
The  urea  is  built  up  in  the  liver,  the  hippuric  acid  in  the 
kidney,  and  the  peptids  in  the  growing  tissues. 

The  enzymes  and  the  substrate  they  work  upon  are  in 
close  chemical  relationship.  E.  Fischer  has  illustrated 
it  by  saying  that  enzyme  and  sugars  are  mutually  related 
to  one  another  in  the  same  way  as  a  key  to  the  lock  which 
it  alone  can  open.  Both  the  chemical  configuration  and 
the  steric,  the  atomic  relation  in  space,  form  the  lock. 
Compounds  resembling  one  another  in  both  features  are 
opened  by  the  same  key.  Erepsin,  and  in  a  lower  degree 
trypsin,  dissociate  all  peptids,  because  in  them  all  the 
amino-acids  are  linked  in  the  same  way  as  in  the  glycyl-gly- 
cine. We  may  regard  all  peptids  as  derived  from  glycyl-gly- 
cine by  substitution  of  an  H  by  a  radical  like  C4H9  (leucine), 
or  C7H8  (tyrosine),  etc.  The  dissociation  always  takes 
place  at  the  already  existing  locus  minoris  resistentia, 
common  to  all  peptids,  peptones,  and  albumins.  In  the 
case  of  the  peptids  and  proteins,  the  action  or  non-action 
of  enzymes  can  be  deduced  from  the  known  chemical 

1  O.  Schmiedeberg:    Archiv  f.  exper.  Path.  u.  Phar.,  14  (1851). 

2  E.  Abderhalden  and  Y.  Teruuchi:  Zeitschr.  f.  physiol.  Chem., 
49  (1906);   O.  Cohnheim:    ibid.,  52  (1907). 


50  ENZYMES 

structure,  and  we  understand  also  why  the  sucroclastic 
enzyme  of  yeast,  the  zymase,  ferments  only  the  four 
sugars  resembling  one  another  and  not  the  other  artificial 
hexoses  with  greater  differences  in  the  other  C-atoms. 
But  we  do  not  understand  why  the  same  enzyme,  invertin, 
which  I  have  said  dissociates  a-methylglucoside,  splits 
up  also  cane  sugar,  and  why  lactase  decomposes  both  the 
glucosides  and  a  compound  so  widely  different  as  milk 
sugar.  The  third  enzyme  affecting  disaccharides,  e.g., 
maltase,  attacks  only  maltose,  and  none  of  the  glucosides 
synthetically  obtained.  It  suggests  to  us  that  our  chemical 
formulae  written  only  on  the  plane  of  the  paper,  do  not 
represent  all  the  space  relations  of  the  molecular 
structure. 

We  can  make  observations  on  the  fate  of  some  aromatic 
amino-acids  in  metabolism.  We  shall  see  later  that  with 
great  probability  all  dissociations  in  the  body  can  be 
ascribed  to  enzymes,  that  metabolic  enzymes  oxidize 
food,  and  change  food  without  oxidation.  If  this 
suggestion  is  right,  we  can  draw  conclusions  from  what 
takes  place  in  metabolism  regarding  the  qualities  of  the 
enzymes  causing  the  changes.  We  observe  that  of  all 
existing  amino-acids,  the  a-amino-acids  alone  disappear 
completely  in  the  human  or  animal  body,  if  given  by 
mouth  or  subcutaneously.  Tyrosine  or  oxyphenylalanine 
and  phenylalanine  are  substituted  alanines,  and  a-alanines. 
Tyrosine,  phenylalanine,  and  all  investigated  compounds 
which  are  substituted  at  the  a-C-atom,  phenyllactic  acid, 
or  compounds  with  a  keto  group  at  this  atom,  are  oxidized 


ENZYMES   AND    OPTICAL   ACTIVITY  51 

completely.  On  the  contrary,  compounds  which  are 
formed  by  substitution  at  the  /9-atom,  are  not  attacked, 
or  they  are  changed  slowly,  in  small  amount  and  in 
successive  stages.  Some  investigators,  like  Knoop,1  have 
thought  them  suitable  for  studying  the  course  of  meta- 
bolism in  the  body;  but  I  think  we  must  be  very  cautious 
in  drawing  conclusions  from  these  abnormal  compounds. 
If  we  cast  a  glance  at  the  formulas  generally  adopted,  we 
must  suppose  that  a-  and  /^-phenylalanine  closely  resemble 
each  other,  and  further,  that  the  differences  must  be 
greater  between  a-  and  /?-isocapronic  acids  than  between 
the  oi-aminoisocapronic  acid  and  aminolactic  acid. 
Nevertheless  the  same  enzyme  attacks  both  the  amino- 
acids  and  leaves  untouched  the  /?-acids.  We  must  sup- 
pose that  the  same  benzene-nucleus,  C6H6,  is  yielded  by 
phenol,  benzene,  or  phenylalanine.  And  if  we  use  only 
inorganic  chemical  means,  we  find  no  great  difference, 
but  if  we  use  enzymes  as  reagents,  we  see  that  benzene 
and  phenol  pass  unchanged  through  the  body  and  that 
only  in  phenylalanine  is  the  aromatic  cycle  broken.  The 
enzymes  exhibit  to  us  differences  not  only  in  the  side- 
chain,  but  in  the  cycle  itself.  We  shall  see  later  that  we 
must  probably  distinguish  between  the  combination  and 
oxidation  in  the  body,  which  sets  free  energy,  and  the 
slow  conversion  without  combustion,  which  is  used  for 
special  functions.  The  enzymes  which  produce  the  first 
effects,  attack  at  once  the  whole  molecule,  and  they  enable 

1  F.  Knoop:    Hofmeister  Beitr.,  6  (1905). 


52  ENZYMES 

us  thus  to  understand  the  intimate  structure  of  the  mole- 
cule more  thoroughly. 

Not  all  enzymes  are  so  specific  as  the  proteolytic  or 
the  sucroclastic  enzymes  already  mentioned.  It  seems 
that  proteins  and  poly-  and  disaccharides  are  never  burned 
by  animals,  plants,  or  bacteria,  without  being  decomposed 
into  monosaccharides  and  amino-acids.  Cane  sugar  and 
milk  sugar  entering  our  body  without  intervention  of  the 
alimentary  canal,  are  eliminated  quantitatively.  Proteins 
which  are  not  previously  dissociated  are  protected  against 
bacteria.  Yeast  fed  on  pentoses,  or  on  hexoses  other 
than  the  four  I  have  mentioned,  starves  as  if  not  fed  at 
all.  But  the  tissue  enzymes  mediating  the  metabolism 
of  the  higher  animals  do  not  show  so  great  a  difference. 
If  we  feed  a  man  or  a  dog  with  pentoses  or  strange  hexoses, 
he  eliminates  them  in  the  urine,  but  not  all  that  was 
absorbed.  The  rest  disappears;  it  must  be  burned  in 
metabolism.  Amino-acids  and  peptids  optically  opposite 
to  the  natural,  are  eliminated  in  part;  another  part  is 
utilized  in  the  body.1 

The  natural  compounds  are  preferred  by  the  metabolic 
enzymes,  and  are  completely  burned  up,  but  these  enzymes 
can  also  attack  other  substances.  The  optical  antipodes 
of  peptids,  given  in  small  quantity,  can  even  disappear 
completely  in  the  organism  of  the  dog  or  rabbit.  Thus 
we  see  that  men  burn  alkaloids,  medicaments,  and  other 
artificial  substances  for  which  they  never  can  produce 

1  E.  Abderhalden,  P.  Bloch,  and  P.  Rona:  Zeitschr.  f.  physiol.  Chem., 
52  (1907). 


ENZYMES   AND   OPTICAL  ACTIVITY  53 

enzymes.  The  enzymes  of  metabolism  have  not  a  com- 
plete specificity,  but  a  relative  one.  These  keys  open  locks 
only  more  or  less  resembling  the  locks  for  which  they  are 
destined.  And  if  we  follow  the  series  of  living  things 
to  the  opposite  end,  to  the  simplest  organism,  we  find  some 
bacteria  which  live  on  almost  any  organic  material.  The 
natural  sugars  and  the  natural  amino-acids  are  preferred 
even  by  them,  but  they  are  not  necessary.  Pfeffer l 
states  that  they  are  extraordinarily  comprehensive  in  their 
activities,  that  is  to  say,  their  enzymes  must  open  almost 
every  kind  of  lock. 

It  will  be  seen  later  on,  that  these  last  enzymes  have 
not  been  thus  far  isolated.  The  enzymes  which  are  well 
known  are  specific,  and  their  relation  to  the  optically 
active  compounds  throws  light  on  the  chemistry  of  the 
enzymes  themselves.  The  enzymes  must  be  optically 
active  compounds.  E.  Fischer  has  discovered  that  the 
two  stereoisomeric  components  of  a  substance  show 
differences  only  in  their  relations  to  optically  active  com- 
pounds, and  not  to  other  substances.  It  often  happens 
that  the  inactive  or  racemic  form  has  quite  other  properties 
than  those  of  the  two  active  forms.  There  are  deviations 
in  melting-point,  solubility,  taste,  and  other  properties, 
but  the  two  active  components  resemble  each  other  in 
all  ways,  except  in  their  rotatory  power  and  in  their 
combinations  with  other  active  bodies.  For  instance, 
the  two  optically  active  compounds  of  benzoyl-aspartic 

1  W.  Pfeffer:    "Pflanzenphysiologie,"  vol.  i,  p.  349. 


54  ENZYMES 

acid  and  their  salts  with  sodium  or  potassium  are  alike 
except  in  rotatory  power,  but  the  salts  of  this  acid  com- 
bined with  optically  active  bases,  such  as  the  components 
of  brucine,  strychnine,  or  quinine,  show  such  differences 
in  solubility  as  to  enable  us  to  separate  the  two  salts  from 
each  other.  It  seems  to  be  a  general  rule,  that  only 
combinations  of  two  optically  active  compounds  differ 
in  this  way,  and  we  must  conclude,  therefore,  that  the 
proteolytic  and  sucroclastic  enzymes  contain  an  un- 
symmetrical  carbon-atom. 


CHAPTER  VII 

Mode  of  Action  of  Enzymes 

The  close  relationship  between  enzymes  and  their 
substrate  mentioned  in  the  last  chapter,  brings  evidence 
that  the  enzymes  enter  into  a  chemical  combination  with 
the  molecules  they  work  upon.  Another  proof  of  this  is 
afforded  by  the  power  of  the  substrate  to  protect  enzymes. 
You  have  heard  how  easily  the  enzymes  undergo  destruc- 
tion or  loss  of  power  by  heat,  by  chemical  processes,  or 
spontaneously.  Kiihne  and  Biernacki 1  observed  that 
proteins  accompanying  trypsin  in  solution  increase  its 
stability.  In  a  purified  solution  of  trypsin  poor  in  protein, 
the  enzyme  loses  its  power  at  400  to  450  C.  in  one  hour, 
while  the  natural  pancreatic  juice  or  a  fresh  extract  of  the 
gland  rich  in  nucleoprotein  can  be  heated  for  one  hour 
at  550  C.  without  damage,  and  is  destroyed  only  at  6o° 
to  650  C.  The  purer  the  enzyme  the  sooner  does  it  lose 
its  activity.  Extracts  of  leucocytes  contain  a  very  small 
quantity  of  trypsin  or  of  a  proteolytic  enzyme  resem- 
bling trypsin,  and  besides  that  they  contain  protein  in 
much  greater  amount.  An  extract  of  leucocytes  can 
be  heated  to  560  C,  and  the  enzyme  resists  formalde- 
hyde, mercuric  chloride,  or    normal    sodium  hydroxide.2 

1  E.  Biernacki:  Zeitschr.  f.  Biol.,  28  (1891). — K.  Mays:  Zeitschr. 
f.  physio!.  Chemie,  38  (1903). 

2  G.  Jochmann  and  G.  Lockemann:    Hofmeister's  Beitrage,  11  (1908). 

55 


56  ENZYMES 

Pepsin  !  undergoes  rapid  destruction  when  separated  from 
the  nucleoprotein  present  in  natural  gastric  juice.  The 
protective  power  of  the  proteins  can  be  explained  in  a 
more  specific  way;  proteins  neutralize  acids,  alkalies, 
formaldehyde,  mercuric  chloride,  and  other  substances 
that  might  precipitate  and  damage  the  enzyme.  But  the 
increased  resistance  of  the  proteolytic  enzymes  to  heat  in 
the  presence  of  proteins  cannot  be  satisfactorily  explained 
in  this  way. 

Sucroclastic  enzymes  are  likewise  protected  by  sugars. 
O' Sullivan  and  Thompson2  found  that  without  cane 
sugar,  invertin  is  almost  wholly  destroyed  by  heating  to 
500  C,  and  to  a  great  extent  by  heating  to  450  C.  When 
cane  sugar  is  present,  it  can  be  heated  to  6o°  C.  without 
losing  any  activity  at  all,  and  on  heating  to  700  C,  it  loses 
only  a  part  of  its  power.  If  we  add  sugar  to  a  solution  of 
zymase,  the  latter  is  capable  of  causing  the  production  of 
carbon  dioxide 

At    6°  C,  during  10  days 

"   22°  C,        "         4     " 

"  35°  C.,       "         1  day 

But  if  we  try  to  preserve  the  press- juice  of  yeast  with- 
out sugar,  we  find  that  the  contained  zymase  loses  its 
activity 

At    o°  C,  in  2  days. 
200  C,  '     1  day. 

1  J.  P.  Pawlow:  Nagel's  "Handbuch,"  vol.  2  (1906);  "Arbeit  der 
Verdauungsdriisen,"  1898. 

2  O'Sullivan  and  Thompson:     Journ.  Chem.  Soc,  1890. 


MODE    OF   ACTION   OF   ENZYMES  57 

For  other  enzymes  no  evidence  on  this  point  has  been 
secured,  but  from  the  fact  that  in  natural  pancreatic  juice 
steapsin  and  nuclease  undergo  deterioration  in  a  very 
short  time,  while  in  the  intestine  the  digestion  of  fat  and 
nucleic  acid  continues  for  many  hours,  we  cannot  doubt 
that  the  law  of  protection  by  the  substrate  holds  good  for 
them  also.  The  enzyme  is  changed  by  the  substrate  it 
works  on,  and  the  solution  does  not  contain  the  same 
enzymic  compound  as  the  pure  solution,  but  it  contains 
another  chemical  compound  of  different  properties,  and 
this  compound  must  be  an  enzyme-protein,  an  enzyme- 
sugar,  etc.  It  is  a  chemical  combination  of  enzyme  and 
substrate.  The  higher  resistance  is  an  evidence  of  the 
existence  of  such  combinations. 

Further  evidence  of  the  combination  of  enzyme  and 
substrate  is  seen  in  the  antitryptic  action  of  serum  albumin 
found  by  Hahn  '  and  explained  by  Hedin.2  A  tryptic 
solution  digests  fibrin,  but  it  cannot  do  so  if  serum  or 
serum  albumin  is  added.  The  trypsin  is  then  bound  by 
the  serum  albumin.  The  serum  albumin,  which  is 
dissolved  with  difficulty  by  trypsin,  takes  away  the  enzyme 
from  the  easily  digestible  fibrin,  and  thus  the  digestion  of 
fibrin  is  checked,  i.e.,  the  enzyme  is  side-tracked. 

In  other  cases,  the  true  chemical  combination  of  enzymes, 
particularly  of  the  proteolytic  enzymes,  with  the  substrates 
is  not  so  clear,  because  the  chemical  combination  is 
complicated  by  the  physical  adsorption  above  mentioned. 

1  M.  Hahn:     Berlin,  klin.  Wochenschr.,  1897. 

2  S.  G.  Hedin:    Zeitschr.  f.  physiol.  Chem.,  52  (1907). 


58  ENZYMES 

If  we  put  fibrin  in  a  solution  of  trypsin  at  zero,  and  observe 
that  the  fibrin  takes  up  all  of  the  trypsin,  we  must  suppose 
— for  the  reasons  above  stated — a  loose  combination  be- 
tween the  protein  and  the  trypsin.  But  fibrin  takes  up 
sucroclastic  enzymes,  hormons,1  etc.,  and  trypsin  is  com- 
pletely taken  up  by  animal  charcoal,2  and  to  a  certain 
extent  by  siliceous  earth.2  The  relations  are,  therefore, 
not  entirely  clear. 

Likewise,  enzymes  enter  into  combination  with  dissocia- 
tion-products. It  is  an  old  observation  that  the  substances 
produced  by  the  action  of  an  enzyme  exert  a  retardation 
of  that  action.  Experimentally,  the  fact  is  of  the  highest 
importance,  because  such  retardation  by  its  own  activity 
is  the  reason  that  the  digestion  in  the  alimentary  canal 
takes  place  under  conditions  very  different  from  those 
which  we  obtain  during  artificial  digestion  in  flasks  and 
beakers.  Trypsin  and  erepsin  do  not  normally  dissociate 
the  protein  in  a  flask,  but  do  in  the  intestine;  the  resulting 
amino-acids  are  immediately  absorbed,  and  the  enzymes 
not  passing  the  intestinal  wall  can  freely  attack  new  mole- 
cules of  protein.  In  the  tissues,  the  structure  of  the  cells 
and  the  circulation  of  the  blood  favor  the  action  of  enzymes 
in  a  similar  fashion.  The  diastase -splits  the  glycogen 
and,  immediately  thereafter,  the  diastase  and  the  sugar 
formed  by  it  are  separated  from  each  other.  Under  these 
conditions  a  comparatively  small  quantity  of  enzymotic 


1  O.  Cohnheim:     Arch,  des  Sciences  biolog.  de  St.  Peters bourg  (n 
Suppl.),  (1904)- 

2  S.  G.  Hedin:    Zeitschr.  f.  physiol.  Chem.,  52  (1907). 


MODE    OF   ACTION    OF   ENZYMES  59 

fluid  is  able  to  dissolve  a  relatively  large  amount  of  the 
substance  it  splits.  Lea x  has  compared  the  digestion 
of  fibrin  by  trypsin  in  a  flask  and  in  a  dialyzer  tube  under 
otherwise  the  same  conditions  and  found,  after  six  hours, 
a  residue  in  the  dialyzer  tube  of  i  gm.,  in  the  flask  2.5 
gra- 
in another  experiment  he  mixed  saliva  and  10  gm.  of 
dextrin,  and  afterward  he  found  0.4  gm.  recoverable 
dextrin  in  the  dialyzer  tube,  and  in  the  flask  2.5  gm. 

He  unfortunately  used  weak  solutions  of  trypsin.  With 
stronger  solutions  the  results  would  be  still  more  striking. 
In  vitro,  peptic  digestion  produces  chiefly  proteoses,  and 
peptones  in  small  amount.  In  the  living  stomach, 
Tobler 2  found,  under  really  normal  conditions,  twenty 
per  cent  proteoses  and  eighty  per  cent  peptones.  I  have 
studied  peptic  digestion  in  dialyzer  tubes,  and  was  able, 
in  nineteen  hours,  almost  completely  to  dissociate  90 
grammes  of  meat -protein  into  peptone.  In  such  experi- 
ments, we  are  therefore  able  to  avoid  a  good  deal  of  the 
retardation  above  referred  to. 

But  what  is  the  reason  of  the  retardation?  In  the 
case  of  those  enzymes  that  act  as  catalyzers,  which  acceler- 
ate only  one  of  the  two  reactions  occurring  without  them, 
and  which  move  the  equilibrium  point,  the  retardation 
can  be  explained  easily.  The  accumulation  of  the  pro- 
ducts of  dissociation  increases  one  side  of  the  equation, 
and  when  the  equilibrium  point  is  approached,  the  reaction 

1  A.  S.  Lea:    Journal  of  Physiology,  n  (1890). 

2  L.  Tobler:     Zeitschr.  f.  physiol.  Chemie,  45  (1905). 


60  ENZYMES 

proceeds  more  and  more  slowly.  But  as  I  have  already 
stated,  the  theory  of  the  catalytic  nature  of  enzymes  holds 
good  only  for  fats  and  fat-splitting  enzymes,  and  the 
explanation  just  stated  does  not  apply  to  the  retardation 
observed  with  proteolytic  and  sucroclastic  enzymes,  be- 
cause the  enzymotic  reactions  are  retarded  and  checked 
not  only  by  their  own  dissociation  products,  but  by  other 
products  resembling  the  substrate  and  dissociation- 
products  in  chemical  and  steric  configuration.  The 
ptyalin  of  the  saliva  decomposes  starch  and  produces 
maltose,  but  the  retardation  is  exerted  not  only  by  maltose, 
but  also  by  glucose,1  which  does  not  appear  in  the  equation 
expressing  the  reaction.  Further  evidence  for  this  has 
been  advanced  by  Abderhalden  and  Gigon.2  They 
allowed  the  proteolytic  enzyme  of  yeast  to  work  on  the 
optically  active  peptid,  glycyltyrosine.  The  enzyme  splits 
the  glycyltyrosine  into  glycocoll  and  tyrosine,  according  to 
the  equation: 

H2NCH2CONHCH(C7H6OH)COOH  +  H20  = 
H2NCH2COOH  +  H2NCH(C7H6OH)COOH. 

The  equation  contains  only  glycocoll  and  tyrosine,  and 
if  the  law  of  equilibrium  holds  for  enzymes,  we  must  ex- 
pect that  only  glycocoll  and  tyrosine  would  retard  the 
reaction.  But  Abderhalden  and  Gigon  have  found  that 
glycocoll,  which  contains  no  symmetrical  carbon  atom, 
exerts  no  retardation,  but  all  other    active  amino-acids, 

1  J.  Cohnheim:    Virchow's  Archiv,  28  (1863). 

2  E.  Abderhalden  and  A.  Gigon:    Zeitschr.  f.  physiol.  Chem.,  53  (1907) . 


MODE    OF   ACTION   OF   ENZYMES  61 

serine,  alanine,  leucine,  tryptophane,  etc.,  do.  Thus  the 
accumulation  of  end-products  cannot  explain  the  retarda- 
tion, because  the  other  amino-acids  are  not  end-products 
of  the  reaction. 

The  only  explanation  I  can  give  is  that  the  enzymes 
combine  with  their  dissociation-products  just  as  they 
do  with  their  substrates.  As  active  compounds,  they 
react  only  with  active  compounds,  but  they  join  with  all 
compounds  of  the  corresponding  steric  and  chemical 
configuration.  A  further  proof  of  this  is  that  trypsin  is 
protected  against  heat  by  peptones  and  amino-acids  in 
the  same  way  as  by  proteins.1 

I  suggest,  for  instance,  that  invertin  is  united  with  cane 
sugar  in  a  loose  chemical  combination.  The  molecule 
of  enzyme-cane-sugar  and  the  enzyme  can  undergo  com- 
bination with  a  new  molecule  of  cane  sugar,  but  it  can 
also  be  abstracted  by  one  of  the  newly-formed  sugars, 
glucose,  or  levulose.  The  retardation  is  not  governed  by 
the  law  of  equilibrium,  but  by  the  law  of  division.  The 
enzyme  can  be  side-tracked  in  the  same  way  by  either  the 
substrate  or  the  dissociation  products. 

If  we  picture  to  ourselves  a  chemical  image  of  the  mode 
of  action  of  enzymes,  we  may  consider  that  invertin  enters 
the  molecule  of  cane  sugar,  and  that  this  new  molecule 
becomes  unstable  and  separates  into  two  smaller  molecules 
resembling  the  greater  one,  and  also  able  to  combine  with 
the  enzyme.     This  would  be  a  strictly  chemical  explana- 


1  E.  Biernacki:     Zeitschr.  f.  Biol.,  28  (1891). 


62  ENZYMES 

tion  of  what  occurs  during  enzymotic  action,  and  we 
know  of  other  analogous  chemical  reactions  as,  for  instance, 
those  in  which  a  chemical  compound  of  great  stability  is 
converted  into  a  more  unstable  one  by  introducing  an 
amino-group  into  the  molecule. 

But,  as  a  matter  of  fact,  the  relations  are  probably  more 
complicated,  because  the  ferments  are  colloids,  that  is  to 
say,  we  are  not  dealing  with  reactions  occurring  in  a 
homogeneous  medium,  but  with  substances  not  really 
dissolved,  but  acting  upon  dissolved  compounds.  Meltzer 1 
refers  to  the  organization  of  ferments,  which  implies  not 
a  simple  chemical  arrangement  inside  of  the  molecule, 
but  a  complex  which  resembles  the  structure  of  living 
protoplasm,  and  consists  of  a  close  connection  cf  fluids 
and  solids  forming  a  physical  rather  than  a  chemical  unit. 

1  S.  J.  Meltzer:     Amer.  Journ.  of  Physiology,  1909. 


CHAPTER   VIII 


Antiferments 


Some  authors l  have  described  compounds  which 
prevent  enzymotic  action  in  a  specific  way,  just  as  anti- 
toxins bind  and  check  toxins.  They  have  met  with  great 
difficulties  in  attempting  to  give  convincing  proofs  for 
this  suggestion.  The  action  of  pepsin  is  disturbed  by 
neutralizing  the  acid  or  by  preventing  the  swelling  of  the 
fibrin.  The  action  of  trypsin,  diastase,  and  steapsin  is 
retarded  by  the  accumulation  of  the  dissociation  products, 
and  the  action  of  all  enzymes,  by  the  addition  of  the 
substrate,  or  adsorption  by  coagulating  proteins,  or 
spontaneous  loss  of  power.  Every  substance  or  every 
condition  which  increases  the  time  of  conversion,  or  has 
a  harmful  influence  on  the  enzyme  or  on  the  conditions  of 
enzymotic  action,  appears  to  us  as  an  antiferment.  Some 
authors  2  have  injected  solutions  of  enzyme  into  the  veins 
of  the  rabbit,  and  have  observed  that  the  blood  of  the 
rabbit  then  yields  a  precipitin,  that  is  to  say,  a  substance 
that  gives  a  precipitate  when  added  to  the  injected  solution. 
In  some  cases  enzymotic  action  in  the  solution  is  prolonged 
or   diminished   under   the    circumstances.     But    the    ex- 

1  E.  Weinland:    Zeitschr.  f.  Biol.,  44  (1902). 

2  J.  Morgenroth:     Zentralblatt  f.  Bakteriol.,  1899. — H.  Sachs,  Fort- 
schr.  d.  Medizin,  20  (1902). 

63 


64  ENZYMES 

periments  are  not  conclusive  because  the  solutions  always 
contained  proteins,  and  these  alone  would  give  rise  to  the 
formation  of  a  precipitin,  and  the  precipitate  formed  by  it 
in  the  solution  might  withdraw  the  enzyme  by  adsorption. 
Hahn  and  Hedin  (see  above)  have  observed  that  the  blood 
serum  of  mammals,  and  to  a  less  degree  the  white  of  egg, 
retards  or  prevents  the  action  of  trypsin,  and  some  authors 
have  spoken  of  the  antiferment  present  in  the  serum.  I 
have  already  given  a  clear  explanation  of  the  phenomenon 
described  by  Hedin;  the  serum  albumin  as  a  slowly 
digestible  protein  appropriates  the  trypsin,  and  diverts  it 
from  the  easily  digestible  fibrin,  and  thus  protects  the 
latter  from  action.  Weinland  observed  that  the  mucous 
membrane  of  the  stomach  and  the  small  intestine,  and 
the  round  worm  frequently  present  in  the  intestine,  yield 
substances  that  retard  or  prevent  the  action  of  pepsin  or 
trypsin.  He  suggested  that  these  results  were  due  to  anti- 
ferments  which  have  a  great  biological  importance,  because 
they  protect  the  worms  and  the  digestive  organs  against 
digestion,  fei  the  case  of  antipepsin  there  can  be  little 
doubt  that  Weinland' s  antipepsin  is  a  protein  or  a  salt 
which  neutralizes  the  hydrochloric  acid,  and,  also,  in 
part,  a  neutral  salt,  which  prevents  the  swelling  of  fibrin 
in  the  hydrochloric  acid.  In  the  case  of  the  intestine  and 
the  worms,  Weinland' s  view  is  not  disproved,  but  there 
are  some  strong  objections  to  it.  Both  the  mucous 
membrane  and  the  body  of  the  worms  contain  proteins 
less  digestible  than  the  fibrin,  which  must  divert  the 
trypsin  from  the  fibrin.     It  is  remarkable  that,  according 


ANTIFERIVENTS  65 

to  Hamburger  and  Hekma,1  the  intestinal  juice  can  like- 
wise disturb  the  digestion  of  fibrin,  although  it  is,  of 
course,  absurd  to  imagine  the  secretion  of  an  antitrypsin 
in  the  intestine. 

Extracts  of  the  small  intestine  also  yield  enterokinase, 
the  activator  of  the  trypsin.  When  we  add  increasing 
quantities  of  intestinal  extract  to  a  given  quantity  of 
pancreatic  juice  the  digestive  power  is  not  proportionately 
increased.  The  first  small  quantities  accelerate,  the 
later  additions  retard,  the  action  of  the  trypsin. 

It  seems  to  me  that  the  evidence  does  not  permit  us 
to  speak  of  specific  antiferments. 

1  J.  H.  Hamburger  and  E.  Hekma:  Journ.  de  Physiologie  et  de  Pa- 
thologie  generate,  1902. 

5 


CHAPTER  IX 

Specificity  of  Enzymes 

It  was  a  question  debated  for  a  long  time  whether  the 
individual  enzymes  occurring  in  different  animals  and  in 
plants  are  identical  or  not.  The  proteolytic  enzymes 
ought  to  be  different  in  different  animals;  for  instance, 
fishes  ought  to  have  another  pepsin  than  mammals,  the 
trypsin  of  the  dog  and  the  ox  should  be  different,  etc. 
But  we  must  make  the  same  objections  as  in  the  case  of 
the  antiferments,  that  the  small  differences  in  the  action 
of  enzymes  can  be  explained  completely  by  the  slight 
differences  in  the  environment,  in  the  method  of  obtaining 
of  the  enzymes,  etc.  Until  we  can  isolate  the  enzymes  as 
chemically  pure  compounds,  we  have  no  means  of  answer- 
ing these  questions  with  certainty,  but  in  all  probability 
the  enzymes  having  similar  actions  are  the  same.  The 
cells  of  our  salivary  glands,  the  protoplasm  of  yeast,  and 
the  germinated  seeds  of  plants  produce  for  the  same 
purpose,  i.e.,  the  digestion  of  starch,  the  same  compounds: 
diastase  and  maltase. 


66 


CHAPTER  X 

Zymogens  and  Activators 

Some  enzymes  are  produced  by  the  cells  in  such  form 
that  they  do  not  need  further  supplement  to  render  them 
active.  This  is  the  case  with  all  enzymes  which  dissoci- 
ate poly-  and  disaccharides,  such  as  diastase,  maltase, 
invertin,  and  lactase.  An  exception  seems  to  be  the 
glycolytic  enzyme  of  the  muscles,  which  appears  to  require 
an  activator  derived  from  the  pancreas.  Other  enzymes 
independent  of  such  aids  are  erepsin  and  the  nucleases. 
The  remaining  enzymes  are  formed  in  the  cells,  and  are 
secreted  from  the  cells  in  an  inactive,  incomplete  form. 
They  meet  with  other  chemical  substances  and  combine 
with  them  to  form  the  complete  enzyme  which  is  then 
capable  of  dissociating  the  appropriate  substrate.  We 
know  the  following  examples  of  such  enzymes: 

i .  The  pepsin  of  the  stomach  is  produced  and  secreted 
by  the  chief  cells  of  the  gastric  glands  as  a  zymogen,  the 
pepsinogen.  Griitzner  x  found,  in  1874,  that  extracts  of 
the  mucous  membrane  of  the  stomach,  in  activity  or  at 
rest,  contain  a  substance  which  is  not  pepsin,  but  yields 
pepsin.  It  therefore  received  the  name  pepsinogen. 
Pepsin    is    destroyed    by    alkalies,    and,    according    to 

1  P.  Griitzner  and  M.  Ebstein:      Pfluger's  Arch.,  8  (1874). 

67 


68  ENZYMES 

Langley,  also  by  carbon  dioxide;  but  pepsinogen  is  not. 
Because  the  same  glands  contain  parietal  or  ovoid  cells 
secreting  the  hydrochloric  acid,  the  secreted  gastric  juice 
always  contains  pepsin.  The  acid  changes  the  pepsinogen 
and  is  also  necessary  for  the  action  of  the  pepsin  formed 
from  it.  We  do  not  know  how  the  enzyme  and  acid  are 
associated.  The  conversion  of  pepsinogen  into  pepsin  is 
apparently  not  reversible. 

2.  Trypsinogen  and  Enterokinase.  As  Heidenhain * 
found  in  1875,  the  extracts  of  the  pancreas  of  the  dog  and 
the  pig  contain  trypsin  in  small  amount;  the  quantity 
increases  if  we  first  treat  the  extracts  with  acetic  acid  and 
then  establish  the  correct  alkaline  reaction.  Kiihne 2 
observed  that  even  the  dried  pancreas  powder  yields 
trypsinogen,  and  Pawlow '  showed  that  the  secreted 
pancreatic  juice  often  contains  no  trypsin,  but  trypsinogen, 
and  that  this  trypsinogen  is  converted  into  trypsin  by 
enterokinase,  a  compound  produced  by  the  mucous 
membrane  of  the  duodenum  and  other  parts  of  the  small 
intestine,  and  present  in  the  enteric  juice.  It  is  possible 
that  trypsinogen  and  enterokinase  combine  with  each 
other  and  thus  form  a  new  compound,  the  real  trypsin. 
But  it  is  also  possible  that  the  enterokinase  converts  tryp- 
sinogen into  trypsin.     Both  views  are  supported  by  ex- 


1  R.  Heidenhain:    Pfliiger's  Arch.,  10  (1875). 

2  W.    Kiihne:      "Untersuch.    a.   d.    physiol.    Institut   Heidelberg,"    i 
(1878). 

3  J.  P.  Pawlow:    Nagel's  "Handbuch,"  2  (1906);  Dissertat.  of  Schepo- 
walnikow  (1898);  Lintwarew  (1901);  Sawitsch,  Russki  Wratch,  1  (1902). 


ZYMOGENS   AND   ACTIVATORS  69 

perimental  facts,  but  the  facts  do  not  enable  us  to  give 
a  clear  decision.  Bayliss 1  has  added  enterokinase  to 
trypsinogen,  and  thus  converted  it  into  trypsin.  When 
he  added  some  of  this  solution  to  another  solution  of 
trypsinogen,  the  new  trypsinogen  was  converted  also. 
Bayliss  concluded  that  enterokinase  is  not  consumed  in 
activating  trypsinogen.  It  acts  therefore  like  a  ferment, 
and  he  suggests  that  enterokinase  does  not  itself  enter 
into  the  composition  of  the  trypsin.  Observations  have 
been  made,  however,  which  support  the  theory  of  the 
linking  together  of  the  two  compounds  to  form  trypsin. 
When  we  add  to  a  solution  of  trypsinogen  increasing 
quantities  of  enterokinase,  the  tryptic  activity  of  the 
mixture  first  increases,  and  then  decreases,  so  that  both 
substances  must  stand  in  a  certain  proportion  to  each 
other  for  the  optimal  efficiency,  and  it  seems  probable 
that  they  are  combined.  The  actual  process  is  not  yet 
clear,  nor  is  it  known  whether  activation  of  trypsinogen  by 
enterokinase  and  the  activation  by  acetic  acid,  as  observed 
by  Heidenhain  and  Kiihne,  are  the  same  or  different 
processes,  so  that  much  has  yet  to  be  learned  regarding 
this  matter. 

3.  Lipase  or  Steapsin  of  the  Pancreas  and  Liver  and 
Bile  Salts.     Nencki,2  Zuntz,3  and  Pawlow  4  observed  that 

1  W.  M.  Bayliss:     Arch,  des  Sciences  Biolog.  de  St.  Peters bourg,  n 
(1904). 

2M.  Nencki:    Arch.  f.  exper.  Path.  u.  Pharm.,  20  (1886). 

3  N.  Zuntz  (and  Ussow) :  Arch.  f.  (Anat.  u.)  Physiol.,  1900. 

4  Lintwarew:     Diss.  St.  Petersbourg,  1901. — J.  P.  Pawlow.    Nagel's 
"Handbuch,"  vol.  ii.  (1906). 


70  ENZYMES 

the  lipolytic  action  of  pancreatic  juice  increases  very  much 
on  adding  bile,  though  the  bile  itself  has  no  lipolytic 
action.  This  case  of  activation  has  been  explained  by 
the  investigations  of  Magnus.1  Magnus  pointed  out 
that  the  bile  salts,  sodium  glycocholate  and  sodium 
taurocholate,  in  the  bile,  are  the  active  compounds,  and 
since  the  salts  prepared  by  synthesis  act  in  the  same  way, 
we  are  sure  that  we  are  not  deceived  by  an  impurity 
adsorbed  by  the  natural  bile  salts.  Magnus 2  further 
showed  that  the  combination  of  the  lipase — he  used  the 
more  stable  lipase  of  the  liver,  probably  identical  with  the 
lipase  of  pancreas — with  the  bile  salts  can  be  separated 
by  dialysis.  The  bile  salts  pass  through  the  parchment 
membrane,  while  the  lipase  remains  within  the  dialyzer 
tube,  and  thus  loses  its  activity.  If  we  add  the  bile  salts, 
the  activity  is  again  restored,  and  the  experiment  can  be 
repeated  several  times.  Lipase  and  bile  salts  join  to- 
gether to  form  a  loose  compound. 

4.  Glycolytic  Enzyme  of  Muscles  and  a  Hormon  of 
the  Pancreas.  If  we  add  glucose  to  a  fresh  water-extract 
of  cat-muscles,  arid  allow  the  extract  to  stand  a  few  hours 
at  380  C,  we  find  the  quantities  of  reducing  sugars  un- 
changed. But  if  we  add  extract  of  pancreas  treated  in 
a  certain  manner,  we  find  after  three  or  four  hours  less 
glucose  than  before.  The  detailed  action  of  the  glycolysis 
is  not  clearly  understood. 

1R.  Magnus:     Zeitschr.  f.  physiol.  Chem.,  48  (1906). — A.  S.  Loeven- 
hart:    Journ.  of  Biol.  Chem.,  2  (1907). 
2  R.  Magnus:    ibid.,  42  (1904). 


ZYMOGENS   AND   ACTIVATORS  71 

5.  Lipase  of  Ricinus-Seeds  and  Acid.  The  germinated 
seeds  of  Ricinus,  the  castor-oil  plant,  contains  a  lipase 
which  hydrolyzes  fats  and  other  esters,  slowly  in  neutral 
solutions,  but  very  rapidly  on  adding  acid.  During  a 
certain  period  of  germination,  an  organic  acid  is  formed, 
and  provokes  the  action  of  the  lipase.1 

6.  Laccase  and  Manganese.  According  to  Bertrand,2 
the  ash  of  laccase,  the  oxidizing  enzyme 'of  the  lac  tree 
of  East  Asia,  always  contains  manganese;  the  activity 
of  laccase  is  associated  with  the  presence  of  manganese, 
and  the  activity  of  an  enzyme  preparation  is  proportional 
to  the  manganese  present. 

7.  A  last  case,  perhaps,  is  the  association  of  zymase 
and  phosphates,3  but  as  I  have  said  before,  we  are  here 
probably  dealing  with  a  mere  improvement  of  reaction. 

The  advantage  of  this  need  of  activation  to  produce 
the  completed  enzyme  seems  clear  in  the  case  of  pepsin 
and  trypsin,  which,  as  zymogens,  cannot  act  within  the 
cells  producing  them.  If  they  could  act  there,  they  would 
destroy  the  protoplasm,  and  if  trypsin  could  work  in  the 
pancreatic  juice,  it  would  hasten  the  destruction  of  other 
enzymes  not  protected  in  the  juice  by  their  substrate. 
The  lipase  of  the  pancreas  does  not  work  without  bile, 
and  we  know  that  bile  dissolves  fatty  acids  and  enables 

1  W.  Connstein,  E.  Hover,  and  E.  Wartenberg:  Ber.  d.  deutsch.  chem. 
Ges.,  35  (1902). — W.  Connstein:  Ergebnisse  der  Physiologie,  iii., 
Biochemie,  1904. 

2  G.  Bertrand,  Compt.  rend.,  124  (1897). 

3  E.  Buchner:  Zeitschr.  f.  physiol.  Chem.,  46  (1905);  Biochem. 
Zeitschr.,  8  (1908). 


72  ENZYMES 

them  to  be  absorbed.  Without  bile,  the  fatty  acids 
not  soluble  in  water  and  not  absorbed,  would  accumu- 
late, and  this  concentration  of  fats  would  then  check 
the  hydrolysis  or  the  process  might  even  be  reversed, 
synthesis  of  fats  resulting. 

In  muscle  the  glycolytic  enzyme  oxidizes  the  sugar 
within  the  fibres,  and  it  is  there  that  the  energy  produced 
by  this  oxidation  is  utilized.  But  since  the  contents  of 
the  muscle  fibre  are  fluid,  it  is  difficult  to  separate  sugar 
and  enzyme,  and  in  this  case  the  requirements  are  met 
by  the  activation  of  the  enzyme  by  acid  as  occasion  may 
demand. 


CHAPTER  XI 

The  Individual  Enzymes 

The  Hydrolytic  Enzymes  of  the  Alimentary  Canal 

This  group  is  the  best  known  of  all,  because  the  enzymes 
are  secreted.  We  can  thus  study  the  natural  juices,  or 
easily  extract  the  enzymes. 

In  man  and  the  higher  animals,  the  following  fourteen 
or  eighteen  enzymes  are  known: 

Diastase  of  the  saliva. 

Diastase  of  the  pancreas. 

Maltase  of  the  small  intestine. 

Lactase  of  the  small  intestine. 

Invertin  of  the  small  intestine. 

Lipase  of  the  stomach. 

Lipase  of  the  pancreas. 

Lipase  of  the  small  intestine. 

Lecithase  of  the  pancreas.        [Perhaps  identical  with 

Lecithase  of  the  small  intestine. )  the  lipases. 

Pepsin  of  the  stomach. 

Protease,  peptone-splitting,  of  the  pyloric  part  of  the 
stomach. 

Trypsin  of  the  pancreas. 

Erepsin  of  the  small  intestine. 

Arginase  of  the  small  intestine.  )  Perhaps  not  digestive 

Arginase  of  the  liver.  )  enzymes. 

73 


74  ENZYMES 

Nuclease  of  the  pancreas. 

Nuclease  of  the  small  intestine. 

The  large  intestine  produces  no  enzymes. 

The  object  of  digestion  is  to  bring  food-stuffs  into 
solution  and  to  convert  them  into  compounds  which  can 
pass  through  the  intestinal  wall.  All  carbohydrates  are 
converted  into  monosaccharides,  glucose,  levulose,  and 
galactose;  all  proteins  into  amino-acids;  all  fats  into 
fatty  acids  and  glycerin;  lecithin  into  fatty  acids,  choline, 
glycerin,  and  phosphoric  acid;  and  nucleic  acid  into 
pyrimidines,  purines,  phosphoric  acid,  and  carbohydrate. 

For  securing  this  complete  disintegration,  each  class  of 
enzyme  action  is  repeated  two  or  three  times  while  the 
food  passes  along  the  alimentary  tract.  If  one  enzyme 
fails,  the  disintegration  can  be  effected  by  the  following 
one.  We  have  two  diastases  in  the  saliva  and  the  pancre- 
atic juice;  three  lipases;  two  enzymes,  pepsin  and  trypsin, 
dissolving  proteins,  and  three  enzymes  dissociating  them: 
trypsin  and  both  erepsins.  Only  the  enzymes  of  the 
small  intestine  are  simple,  i.e.,  require  no  activator,  and  I 
have  spoken  of  their  exceptional  behavior,  because  they 
seem  to  act  to  some  extent  within  the  cells,  and  attack 
the  compounds  which  pass  through  them. 

Carbohydrate-Splitting  Enzymes 

The  saliva,  the  first  digestive  fluid,  contains  diastase 
or  ptyalin  which  converts  starch  and  glycogen  into  maltose. 
Starch  and  glycogen  are  anhydrides  of  the  sugars,  and 
do  not  give  the  reduction  reactions  of  sugars — the  tests  of 


THE    INDIVIDUAL   ENZYMES  75 

Trommer,  Fehling,  Pavy,  or  Almen-Nylander.  With 
iodine,  starch  gives  a  blue,  and  glycogen  a  red  reaction. 
The  conversion  can  be  followed  by  the  disappeance  of 
these  color  reactions,  or  by  the  appearance  and  increase 
of  the  reducing  power.  The  transition  is  a  very  gradual 
one,  owing  to  the  existence  of  a  number  of  intermediate 
substances,  the  dextrins  and  isomaltose.  The  isomaltose 
alone  is  a  distinct  chemical  individual.  The  dextrins  are 
mixtures  which  have  not  been  separated  into  their  con- 
stituents. Among  them  we  can  distinguish  two  groups 
by  means  of  the  color  reactions  with  iodine:  the  erythro- 
and  the  achroo-dextrins.  In  the  beginning  of  the  con- 
version, starch  loses  its  colloidal  character,  and,  without 
changing  its  chemical  properties,  becomes  readily  soluble 
in  water.  The  different  reactions  with  iodine  are  as 
follows : 

Starch,  blue,  no  reduction. 

Soluble  starch,  blue,  no  reduction. 

Erythrodextrin,  red,  reduction. 

Achroodextrin,  no  color,  reduction. 

Isomaltose,  no  color,  reduction. 

Maltose,  no  color,  reduction. 

The  compounds  or  group  of  compounds  appear  and 
disappear  one  after  another,  but  from  the  beginning  of 
the  conversion  all  dissociation  products  are  present 
simultaneously.  The  real  chemical  nature  of  this  process 
is  not  yet  known.  The  conversion  by  ptyalin  is  the  same 
as  the  conversion  by  dilute  boiling  acids.  The  only 
difference  seems  to  be,  that  the  enzymotic  dissociation  by 


76  ENZYMES 

ptyalin  stops  at  maltose,  while  starch  boiled  with  acids  is 
converted  finally  into  glucose.  The  conversion  of  maltose 
into  glucose  is  effected  by  a  special  enzyme,  maltase,  as 
Musculus  and  von  Mering  *  pointed  out  in  1878.  These 
enzymes,  so  closely  related  in  their  action,  may  be  produced 
in  the  same  cells;  such  is  the  case  in  the  tissues,  blood 
corpuscles,  and  in  plants;  but  they  are  separated  in  the 
alimentary  canal.  Saliva  and  pancreatic  juice  contain 
no  maltase,  and  the  enteric  juice  contains  maltase  but 
no  ptyalin,  or  only  traces.  The  ptyalin  of  the  saliva  is, 
so  far  as  we  know,  identical  with  the  ptyalins  of  the  liver, 
of  the  pancreas,  muscles,  malt,  yeast,  and  other  plants. 

The  mixed  human  saliva  always  contains  a  great  deal  of 
ptyalin,  and  so  does  the  extract  of  the  human  submaxillary 
gland.  The  parotid  saliva  seems  to  be  less  active.  In 
the  pig  and  guinea-pig  also  all  the  salivary  glands  produce 
a  large  amount  of  enzyme;  while  in  the  rabbit  only  the 
parotid  saliva  is  rich,  the  submaxillary  saliva  poor  in 
enzyme.  In  other  animals,  ptyalin  rarely  occurs  in 
saliva.  Neither  the  saliva  of  the  horse  nor  the  extracts 
of  the  salivary  glands  of  the  large  ruminant  animals,  the 
ox  and  sheep,  appear  to  contain  ptyalin.  The  saliva  of 
dogs  and  cats  shows  at  most  an  extremely  feeble  amylolytic 
power,  often  none  at  all;  it  contains  much  less  enzyme  than 
do  the  blood  and  lymph  of  the  same  animals.2     Experi- 

1  F.  Musculus  and  J.  von  Mering-  Zeitschr.  f.  physiol.  Chem.,  2 
(1878). — C.  Hamburger:     Pfluger's  Arch.,  60  (1895). 

*  L.  B.  Mendel  and  Frank  P.  Underhill:  Journ.  of  Biol.  Chem.,  3 
(1907). 


THE   INDIVIDUAL  ENZYMES  77 

ments  on  the  influence  of  different  stimuli  on  the  amount 
of  enzyme  have  been  carried  out  only  on  dogs,  but  these 
animals  are  not  suitable  for  such  investigations. 

The  action  of  ptyalin  begins  in  the  mouth  during  masti- 
cation, and  is  continued  in  the  stomach.  The  action  is 
checked  by  acid,  but  the  gastric  juice  comes  in  contact 
only  with  the  outer  parts  of  the  contents  of  the  cardiac 
end.  It  has  been  pointed  out  by  Ellenberger,1  Cannon,2 
and  Grutzner,3  that  the  inside  of  the  food-mass  lying  in 
the  cardiac  end  of  the  stomach  has  the  slight  alkaline  re- 
action of  the  saliva  which  moistens  the  food.  Thus  under 
normal  conditions  a  great  deal  of  starch  is  dissolved  in 
the  stomach,  and  reaches  the  intestine  as  dextrin  or 
maltose.  Here  it  meets  with  the  pancreatic  juice,  which 
contains  the  same  ptyalin  as  the  saliva.  The  pancreatic 
juice  and  pancreatic  extract  are  rich  in  ptyalin  in  all 
vertebrates  which  have  been  examined;  in  man  and 
other  mammals  ptyalin  is  present  during  the  last  few 
weeks  before  birth. 

We  know  of  a  variety  of  stimulants  for  the  secretion  of 
the  pancreas,  which  induce  the  secretion  of  juices  of 
different  properties. 

i.  The  presence  of  acid  in  the  intestine  calls  forth,  by 
means  of  a  hormon  secretin  of  the  intestinal  wall,  a 
strongly  alkaline  juice  poor  in  solids,  especially  proteins. 

2.  Odor,  sight,  or  the  taste  of  food,  provokes,  by  a 

1  Ellenberger:    Pfliiger's  Arch.,  114  (1906). 

2  W.  B.  Cannon:    Amer.  Journ.  of  Physiology,  9  (1903). 
3P   Grutzner:    Pfliiger's  Arch.,  106,  463  (1905). 


78  ENZYMES 

nervous  mechanism,  the  so-called  psychical  reaction,  a 
secretion  containing  much  protein,  but  little  alkali. 

3.  Fats,  fatty  acids,  and  soaps  provoke  a  secretion 
from  the  duodenum  by  an  unknown  mechanism.  This 
juice  is  still  richer  in  proteins  than  that  occasioned  by  the 
psychical  reaction,  and  is  only  slightly  alkaline.1  Dogs 
fed  with  meat  secrete  a  pancreatic  juice  resembling  the 
pure  secretin-juice,  and  this  is,  according  to  Walther,2 
a  pupil  of  Pawlow,  poor  in  ptyalin.  Dogs  fed  with  milk 
and  bread  secrete  by  a  summation  of  stimuli  a  more  con- 
centrated juice,  which  contains  more  ptyalin.  We  have  no 
evidence  of  other  adaptations  of  the  enzymes  of  the 
pancreas  to  the  properties  of  food. 

Enzymes  inverting  disaccharides  are  limited  to  the 
small  intestine.  It  is  pointed  out  by  Bainbridge,3  Plimmer,4 
and  Ibrahim 5  that  the  pancreas  does  not  produce  lactase 
even  in  new-born  mammals  fed  exclusively  upon  milk. 
Maltase  occurs  in  all  tissues,  while  lactase  and  invertin 
are  produced  exclusively  by  the  mucous  membrane  of 
the  small  intestine.  Cane  sugar  and  milk  sugar  can,  there- 
fore, be  utilized  only  if  they  meet  with  the  enzymes  of  the 
intestine.  As  was  said,  enzymes  probably  attack  disac- 
charides not  only  in  the  lumen  of  the  intestine,  but  to  a 
great  extent  during  the  passage  through  the  cells. 

1  J.  P.  Pawlow:    Nagel's  "Handbuch  der  Physiologie,"  Bd.  ii.,  1906. 

2  A.  A.  Walther:  Arch,  des  Sciences  biolog.  de  St.  Petersbourg,  7(1899). 

3  F.  A.  Bainbridge:     Journ.  of  Physiol.,  31  (1904). 

4  R.  H.  A.  Plimmer:    ibid.,  34  (1906). 

5  J.  Ibrahim  and  L.  Kaumheimer:  Zeitschr.  f.  physiolog.  Chem.,  62 
(1909). 


THE   INDIVIDUAL  ENZYMES  79 

Maltase  is  always  found  in  the  extract  of  the  intestinal 
mucous  membrane,  and  invertin  or  sucrase  is  one  of  the 
enzymes  occurring  first  in  embryonic  life.  In  the  fetus 
of  man,  dogs,  and  cats,  it  can  be  found  earlier  than 
erepsin,  trypsin,  and  ptyalin.1  We  do  not  yet  understand 
what  this  fact  means;  it  is  remarkable  that  we  find  in 
the  stomach  and  in  the  intestine  of  the  fetus  a  reducing 
and  levorotatory  fluid.  Mendel 2  found  invertin  in  pigs 
only  shortly  before  birth,  and  maltase  and  lactase  in  the 
middle  of  fetal  life.  The  human  fetus  produces  lactase 
only  shortly  before  birth.3 

Of  the  inverting  enzymes  the  most  interesting  to-day  is 
lactase,  because  lactase  is  the  only  enzyme  for  which  we 
seem  to  have  evidence  of  adaptation  to  diet,  i.e.,  to  the 
need  of  the  body  during  individual  life.  Milk  sugar 
occurs  only  in  the  milk  of  mammals,  and  it  has  been 
pointed  out  in  recent  years  by  Weinland,4  Bainbridge, 
Plimmer,  Mendel,1  and  others  that  all  young,  sucking 
mammals  produce  lactase,  while  birds,  frogs,  and  inverte- 
brates do  not.  The  presence  of  lactase  in  adult  mammals 
is  questioned.  Weinland,  Mendel,  Bierry,  and  others  have 
come  to  the  conclusion  that,  as  a  rule,  lactase  is  not  found 
in  adult  animals,  such  as  the  pig,  guinea-pig,  rabbit,  dog, 
sheep,  cat,  ox,  or  horse.  Weinland  observed,  however, 
that  dogs  fed  for  some  weeks  with  milk,  or  milk  sugar, 

1  J.  Ibrahim:    Gesellsch.  f.  Kinderheilkunde,  1908. 

2  L.  B.  Mendel  and  P.  H.  Mitchell :  Amer.  Journ.  of  Physiol.,  20  (1907). 

3  J.  Ibrahim:     he.  cit. 

*  E.  Weinland.   Zeitschr.  f.  Biol.,  38  (1899). 


80  ENZYMES 

produce  lactase  again,  and  he  concluded  that  this  was 
adaptation  to  diet.  Further  experiments  carried  out  by 
Plimmer  seem  to  show  that  no  adaptation  occurs.  He 
always  found  lactase  in  the  intestines  of  adult  pigs,  dogs, 
and  cats,  and  failed  to  find  it  in  adult  guinea-pigs,  whether 
fed  with  milk  or  not.  These  discrepancies  are  without 
doubt  occasioned  by  the  great  difficulties  experienced  in 
detecting  and  estimating  galactose  and  glucose,  besides 
the  milk  sugar,  and  by  the  danger  that,  in  extracts  of  the 
intestine  crowded  with  bacteria,  glucose  is  formed  by 
bacteria  and  not  by  the  enzyme.  In  my  opinion,  the 
results  of  Weinland  and  Mendel  are  not  set  aside  by 
Plimmer.  The  occurrence  of  lactase  induced  by  feeding 
milk  sugar  is  the  only  case  of  an  adaptation  of  enzymes 
during  individual  life. 

At  this  point  may  be  mentioned  the  experiments 1 
carried  out  with  the  aim  of  seeing  whether  animals  can 
produce  new  enzymes  not  natural  to  them.  The  results 
were  negative.  Birds  fed  with  milk  sugar  do  not  produce 
lactase,  nor  do  dogs  fed  with  inulin  2  or  polysaccharides 
built  up  from  mannose  and  galactose,  produce  the  corre- 
sponding enzymes  which  occur  in  plants  and  bacteria. 


1  E.  Weinland:    Zeitschr.  f.  Biol.,  47  (1905). 

2  L.  B.  Mendel  and  T.  Taiki:    Journ.  of  Biolog.  Chemistry,  2  (1906). 


CHAPTER   XII 

The  Lipases  or  Steapsins  of  the  Alimentary 

Canal 

The  three  known  lipases  or  steapsins  work  in  the  same 
way;  i.e.,  they  split  neutral  fats  into  fatty  acids  and  glyc- 
erin. They  differ  only  in  the  conditions  of  action,  the 
final  reaction,  the  activation  by  bile,  and  such  secondary 
characteristics.  The  lipase  of  the  stomach  was  discovered 
by  Volhard  l  in  1900.  It  is  secreted  by  the  cells  of  the  gas- 
tric glands,  and  is  found  both  in  the  natural  gastric  juice, 
in  the  contents  of  the  stomach,  and  in  the  extracts  of  the 
glands.  Because  oil  can  produce  under  special  conditions 
the  entrance  of  bile  and  pancreatic  juice  into  the  stomach, 
some  authors  have  believed  that  Volhard' s  lipase  is  the 
pancreatic  lipase  present  in  the  stomach,  but  Volhard  and 
Magnus  and  Laqueur 2  have  found  it  in  the  juice  of 
Pawlow's  so-called  little  stomach,  separated  completely 
from  the  gastric  cavity,  and  inaccessible  to  the  pancreatic 
juice.  Whether  gastric  juice  always  contains  lipase,  or 
whether  the  secretion  occurs  only  in  response  to  a  special 
stimulus,  has  not  been  investigated.  The  gastric  lipase 
is  active  in  a  slightly  acid  medium;  a  stronger  acid  reaction 

1  F.  Volhard:  Miinchener  med.  Wochenschrift,  1900,  141  and  195. — 
Zeitschrift  f.  klin.  Medizin,  42,  414;  43,  397  (1901). — Hofmeister's  Bei- 
trage,  3  (1902);  7  (1905). 

2  E.  Laqueur:    Hofmeister's  Beitrage,  8  (1906) 

6  81 


82  ENZYMES 

checks  its  action.  It  is  to  be  noted  that  fats  retard  the 
secretion  of  gastric  juice  and  thus  lessen  the  amount  of  acid 
in  the  stomach.  The  gastric  lipase  attacks  only  emulsified 
fats,  for  instance  the  fats  of  milk,  and  especially  of  eggs. 
For  estimating  gastric  lipase,  Volhard  uses  egg  yolk. 
There  is  no  evidence  of  the  existence  of  a  zymogen  or 
an  activator.  The  new-born  child  produces  gastric 
lipase.1 

The  pancreatic  lipase  has  been  known  for  a  long  time. 
It  attacks  all  fats,  whether  emulsified  or  not.  It  works 
best  in  neutral  or  slightly  alkaline  reaction.  It  is  more 
unstable  than  other  lipases,  and  perishes  rapidly  in  natural 
pancreatic  juice  or  fresh  water-extracts  of  the  gland;  it 
is  apparently  destroyed  by  trypsin.  Pancreatic  juice 
always  contains  a  certain  amount  of  ready  lipase.  Most 
of  it  occurs  as  a  zymogen,  which  is  made  active  by  bile 
salts;  the  only  activation  completely  explained.  Fats 
stimulating  the  intestinal  wall  cause  pancreatic  juice  and 
bile  to  be  poured  out  at  the  same  time.  The  action  of  the 
pancreatic  steapsin  is  promoted  by  the  bile  salts  in  two 
ways.  The  enzyme  is  activated  by  them,  and  they  dissolve 
the  fatty  acids  set  free  by  the  steapsin  and  enable  them  to 
be  absorbed.2 

The  third  lipase  occurring  in  the  enteric  juice  was 
discovered  by  Pawlow  and    Boldyreff.3     It  attacks   only 

1  J.  Ibrahim  and  T.  Kopez:    Zeitschr.  f.  Biol.,  53  (1908). 

2  B.  Moore  and  D.  P.  Rockwood:    Journ.  of  Physiol.,  21  (1897). 

3  W.  Boldyreff :  Arch,  des  Sciences  biolog.  de  St.  Petersbourg,  11 
(1904). 


LIPASES   OF   THE   ALIMENTARY   CANAL  83 

emulsified  fats.     The  optimum  for  its  action  is  a  slightly 
alkaline  reaction.     It  is  not  activated  by  bile. 

There  can  be  no  doubt  that  all  fats  are  split  by  these 
three  enzymes  and  dissolved  in  water  through  the  agency 
of  bile.  During  passage  through  the  cells  of  the  mucous 
membrane,  neutral  fats  are  again  produced  from  the 
dissociation  products.  Lipases  can  both  build  up  and 
split  fats.  It  depends  upon  the  concentration  and  upon 
the  presence  of  more  or  less  water,  as  to  which  process 
prevails.  The  synthesis  of  fats  in  the  cells  lining  the 
intestine  is  due  to  the  lipase  manufactured  by  these  cells. 
As  we  can  see  microscopically,  very  minute  drops  of  fats 
or  of  fatty  acids  (which  cannot  be  distinguished  optically 
from  each  other)  become  visible  in  the  first  row  of  epithelial 
cells.  During  the  solution  of  these  acids  in  the  watery 
contents  of  the  intestine,  we  do  not  see  them.  On  entering 
the  cells,  they  are  precipitated,  that  is  to  say,  they  are 
separated  from  the  water  and  then  the  synthetic  action 
of  the  intestinal  lipase  begins.  After  synthesis,  fats  lose 
their  solubility  in  water,  and,  as  insoluble  globules,  they 
cannot  pass  the  wall  of  the  endothelial  cells  and  enter  the 
capillaries,  but  must  follow  the  spaces  of  the  reticular 
tissue.  They  are  therefore  conveyed  into  the  lacteal 
radicles  and  the  lymphatic  vessels. 


AJ 


CHAPTER  XIII 


Proteolytic  Enzymes 


The  proteolytic  enzymes,  pepsin,  trypsin,  and  erepsin, 
are  exactly  adapted  to  one  another.  The  proteins  are 
handed  from  one  enzyme  to  the  next  like  bricks  in  the  hands 
of  the  mason.  The  first  passes  the  brick  to  the  second, 
the  second  to  the  third,  etc.  But  while  the  brick  goes 
from  hand  to  hand  without  changing,  the  proteins  are 
divided  into  small  pieces  before  being  useful  to  build  a 
new  body.  The  first  enzyme,  pepsin,  dissolves  all  natural 
proteins,  but  converts  them  only  into  proteoses  and 
peptones;  tfhe  second,  trypsin,  does  not  dissolve  all,  but 
most  proteins,  and  splits  them  and  the  peptones  chiefly 
into  amino-acids;  erepsin  does  not  act  upon  any  natural 
protein,  and  digests  proteoses  but  slowly,  but  it  dissoci- 
ates the  peptones  formed  by  pepsin  much  more  rapidly 
than  does  trypsin;  it  converts  peptones  completely  into 
amino-acids.  Thus  the  complete  disintegration  of  the 
protein  molecule  is  secured,  even  if  gastric  digestion  or  the 
secretion  of  the  pancreatic  juice  is  somewhat  disturbed. 
We  know  in  human  pathology  cases  of  the  so-called  gastric 
achylia,  and  I  have  observed  a  similar  condition  in  the 
dog,1  in  which  the  secretion  of  the  stomach  may  fail  com- 

1  O.  Cohnheim:    Miinchener  med.  Woch.,  1907. 
84 


PROTEOLYTIC  ENZYMES  85 

pletely  without  causing  any  trouble  to  the  patient,  and 
without  reducing  the  availability  of  food. 

Pepsin  acts  in  the  presence  of  hydrochloric  acid.  Kiihne  * 
was  the  first  to  observe  that  pepsin  does  not  form  amino- 
acids,  but  only  peptones.  His  view  has  been  supported 
in  recent  years  by  many  observers.2  No  free  tyrosine  or 
tryptophane  occurs  in  the  stomach  or  in  peptic  digestion. 
Tyrosine  is  nearly  insoluble  in  water,  and  the  so-called 
tryptophane  reaction,  a  violet  color  with  bromine  or 
chlorine  water  and  acetic  acid,  is  given  only  by  the  free 
tryptophane  not  bound  in  the  protein  molecule.  Thus 
the  absence  of  tyrosine  crystals  and  the  negative  result  of 
the  tryptophane  reaction  distinguish  peptic  from  tryptic 
and  other  digestions.  Pepsin  does  not  directly  produce 
the  so-called  peptones,  but  the  transition  is  a  gradual  one, 
and  the  intermediate  products  were  called  albumoses 
by  Kiihne.3  Kiihne  and  most  earlier  investigators,  study- 
ing peptic  digestion  in  vitro  or  in  the  stomach,  believed 
that  the  proteoses  were  the  chief  products  of  pepsin 
digestion.  Now  we  know  from  Tobler,  that  in  the  living 
stomach  peptic  digestion  proceeds  much  more  rapidly, 
and  that  proteoses  can  be  found  early  in  very  small 
quantity  in  the  chyme  leaving  the  stomach.  Even  in 
artificial   digestion,  proteoses  disappear  rapidly  as    de- 

1 W.   Kiihne:     "Untersuch.   a.   d.   physiol.   Institut  Heidelberg,"  ii. 
(1878). — Verhandl.  des  naturh.  Vereins  Heidelberg  (N.  F.),  i. 

2  L.  Tobler:    Zeitschr.  f.  physiolog.  Chemie,  45  (1905). — S.  S.  Salaskin, 
ibid.,  32  (1901);  38  (1903). — O.  Cohnheim:  Miinchener  med.Woch.,  1907. 

3  The  term  proteose,  as  suggested  by  Chittenden,  will  be  used  by  the 
editor  instead  of  albumose. 


86  ENZYMES 

scribed,  if  we  use  dialysis  and  thus  imitate  natural  con- 
ditions. The  proteoses  seemed,  for  many  years,  to  be 
substances  of  the  highest  interest,  and  their  occurrence, 
their  absorption,  their  classification,  and  their  toxicity, 
were  often  and  thoroughly  studied;  we  must  now  say, 
however,  that  their  importance  was  much  exaggerated. 
Of  the  peptones  formed  by  pepsin,  no  chemical  individuals 
seem  as  yet  to  have  been  isolated.  Most  of  them,  but 
apparently  not  all,  give  a  positive,  strong,  biuret  reaction. 

The  secretion  of  gastric  juice  can  be  stimulated  both  by 
nervous  mechanisms  from  the  sense-organs  of  the  head 
and  from  the  intestine,  and  by  a  chemical  mechanism, 
by  a  hormon  produced  in  the  antrum  pylori.  These 
juices  contain,  according  to  Pawlow,  the  same  quantity 
of  pepsin,  but  if  we  add  starch  to  meat,  stimulating 
secretion,  the  concentration  of  pepsin  rises.  The  function 
of  this  action  has  not  been  explained,  but  it  is  remarkable 
that  many  years  ago  SchifT  1  described  peptogenic  sub- 
stances, that  is  to  say,  substances  which,  without  causing 
secretion,  give  rise  to  the  production  of  pepsin  in  cells. 

The  amount  of  hydrochloric  acid  seems  to  be  identical  in 
man,  dog,  and  goat,  but  human  juice  yields  less  pepsin 
than  the  dog's  secretion,  and  thus  dissolves  less  protein. 
Ten  cc.  of  the  contents  of  the  stomach  three  hours  after 
a  test  meal,  contains  12  mgm.  nitrogen  in  the  case  of  man, 
and  30  mgm.  in  the  case  of  the  dog.2 


1  A.  Herzen:    Pfliiger's  Arch.,  84  (1901). 

2  O.   Cohnheim  and   G.   L.   Dreyfus:     Zeitschr.   f.   physiol.   Chemie, 
58(1908). 


PROTEOLYTIC   ENZYMES  87 

All  proteins  are  dissolved  in  the  gastric  juice,  and  the 
casein  of  milk  becomes  solid  in  the  stomach.  It  has  been 
known  for  a  long  time,  that  an  extract  of  the  calf's  stomach, 
called  rennet,  has  a  remarkable  effect  in  rapidly  curdling 
milk,  and  this  property  has  been  utilized  for  thousands  of 
years  in  the  manufacture  of  cheese.  If  a  few  drops  of 
gastric  juice  or  of  gastric  extract  are  added  to  milk,  and 
the  mixture  exposed  to  body  temperature,  the  milk  curdles 
into  a  complete  clot  in  less  than  a  minute.  If  the  action 
is  continued,  the  clot  is  dissolved.  In  1872,  Hammarsten 
showed  that  the  curdling  is  due  to  the  precipitation  of 
casein,  and  that  the  curdling  properties  of  the  gastric 
infusions  are  destroyed  by  boiling.  He  suggested  that  in 
curdling  the  casein  is  converted  into  a  new  compound, 
paracasein,  insoluble  in  water,  and  that  this  conversion  is 
caused  by  a  special  enzyme,  rennin.  This  view  has  been 
generally  accepted  for  over  thirty  years,  although  we  to- 
day have  reasons  for  doubt.  Casein  is  the  only  protein 
precipitated  by  rennin,  and  occurs  only  in  mammals'  milk ; 
nevertheless,  the  alimentary  canal  or  the  tissues  of  birds, 
frogs,  fish,  invertebrates,  bacteria,  and  plants,  all  contain 
considerable  rennin.  I  remember  how  astonished  I  was 
to  meet  with  rennin  in  polyps,  sea-urchins,  and  star-fish, 
which  never  drink  milk.  Pawlow  *  solved  the  riddle; 
he  found  that  rennin  and  pepsin  cannot  be  separated  from 
each  other  and  that  the  amount  of  rennin  and  pepsin  in 

1  J.  P.  Pawlow  and  S.  Parastschuk:  Zeitschr.  f.  physiolog.  Chem., 
42  (1904). — W.  W.  Sawitsch:  ibid.,  55  (1908);  63  (1909). — M.  Jacoby: 
Biochem.  Zeitschr.,  1  (1906). 


88  ENZYMES 

gastric  juices,  gastric  extract,  and  dried  enzyme  powder 
is  always  proportional.  Vernon  '  noted  the  uniformity 
of  rennin  and  trypsin  in  pancreatic  extracts,  and  Pawlow 
and  his  collaborators,  especially  Sawitsch,  were  able  to 
refute  all  objections.  We  cannot  doubt  to-day  the 
identity  of  pepsin  and  rennin,  but  I  think  that  we  must 
go  further  than  Pawlow,  who  believed  that  the  same 
enzyme-molecule  has  two  enzymotic  actions.  Hammar- 
sten  observed  that,  after  curdling,  not  all  the  casein  is 
removed  from  the  solution,  but  that  there  remains  a 
remnant  of  peptone-like  substance;  and  he  suggested 
that,  in  curdling,  the  casein  is  split  into  two  compounds. 
Because  all  proteolytic  enzymes  affect  casein  in  the  same 
way  as  pepsin  does,  it  seems  to  me  that  the  best  explana- 
tion for  all  the  facts  is  that  the  curdling  is  only  the  first 
step  toward  the  dissociation  of  casein.  Rennin  is  there- 
fore not  a  special  enzyme,  but  the  casein  is  disintegrated 
very  easily  by  all  proteolytic  enzymes,  and  the  first  cleavage 
product,  the  first  proteose  of  the  casein,  is  insoluble  in 
acids  and  in  water  containing  lime  salts.  The  misleading 
phenomenon  is  due  to  the  extreme  sensitiveness  of  the 
clotting  reaction,  which  led  to  a  separation  of  the  clotting 
of  casein  from  the  common  action  of  pepsin.  Casein 
is  the  most  easily  digested  of  all  proteins;  it  is  itself  acid 
and  needs  no  further  acid,  and  the  precipitation  of  casein 
is  more  obvious  than  the  beginning  digestion  of  other 
proteins. 

1  H.  M.  Vernon:    Journ.  of  Physiol.,  28  (1903). 


PROTEOLYTIC   ENZYMES  89 

The  stomach,  in  mammals,  is  not  peculiarly  adapted 
to  the  needs  of  milk  digestion,  but  the  mammary  gland 
secretes  a  protein  adapted  in  a  remarkable  way  for  the 
processes  occurring  in  the  stomach.  Fluids  pass  rapidly 
through  the  stomach  without  being  digested.1  The  milk, 
so  rich  in  proteins  and  fats,  must  be  thoroughly  digested, 
and  this  effect  is  obtained  by  changing  milk  to  a  solid 
mass.  Tobler 2  observed  that,  in  the  stomach,  milk  is 
divided  into  two  parts.  Water  and  milk  sugar  leave  the 
stomach  in  a  short  time,  while  casein  and  fats  remain  for 
many  hours  in  the  stomach  and  are  digested  there  slowly 
and  completely.  Clotting  of  milk  is  very  important,  but 
rennet  is  no  special  enzyme;  and  this  fact  is  of  great 
interest,  because  many  investigations  of  the  general  nature 
of  enzymes  and  of  the  laws  of  enzyme-action,  deal  with 
rennet.  If  clotting  is  only  a  short  intermediate  process, 
such  observations  lose  value  as  evidence,  and  we  meet 
here  with  one  of  the  unfortunate  cases  in  which  science 
in  stepping  forward  obliterates  and  renders  useless  the 
hard  and  skilful  work  of  a  whole  generation  of  prominent 
men.  The  clotting  of  milk  and  the  properties  of  the  serum 
have  been  studied  by  biologists  and  chemists  like  Hammar- 
sten,  and  now  his  work  is  set  aside.  Just  as  casein  gives 
rise  to  an  insoluble  proteose,  so  do  occasionally  other 
proteins.  We  can  see  a  precipitation  more  or  less  bulky, 
particularly  on  heating  proteins  with  proportionately 
small  quantities  of  pepsin  and  an  insufficient  quantity  of 

1  O.  Cohnheim:    Miinchener  med.  Wochensch.,  1907. 

2  L.  Tobler:    Gesellsch.  f.  Kinderheilkunde,  vol.  23  (1907). 


90  ENZYMES 

acid,  and  if,  after  a  time,  fresh  pepsin  is  added.  Danilew- 
sky  1  and  his  collaborators,  Sawjalow,  Kurajeff,  and  others, 
have  thought  that  they  were  dealing  with  a  synthetic 
process  of  great  physiological  importance,  the  building 
up  of  protein  from  peptones.  They  gave  to  the  substance 
precipitated  the  name  plastein.  Salaskin,  Bayer,  Levene,2 
and  others  have  shown  that  plastein  is  not  a  protein,  but 
a  proteose-like  body  or  an  impurity,  precipitated  by  the 
acid  or  one  of  the  substances  of  the  extract.  It  most 
resembles  the  hetero-proteose  or  dys-proteose  of  Kiihne 
which  becomes  insoluble  during  peptic  digestion.  If  the 
digestion  continues,  the  plastein  is  dissolved  again  and 
finally  is  converted  into  peptones  like  other  proteoses. 
The  gastric  contents  which  pour  through  the  pylorus  con- 
tain no  plastein,  and  no  conclusive  evidence  has  been 
brought  forward  that  the  protein  absorbed  from  the 
stomach  yields  any  of  this  plastein. 

The  stomach,  or  at  least  a  part  of  the  stomach,  contains 
a  second  proteolytic,  or  better,  peptolytic,  enzyme,  ob- 
served by  Malfatti,3  Bergmann,4  Takamura,5  and  others.6 
The  enzyme,  or  as  most  authors  say,  the  protease,  like 

1  Okunew:  Diss.  St.  Petersbourg,  1895. — Sawjalow:  Pflliger's  Arch., 
85  (1901). — Kurajeff:  Hofmeister's  Beitr.,  1  (1901). — Nurnberg:  ibid., 
4  (1903). — Bayer:  ibid.,  4  (1903). — Salaskin:  Zeitschr.  f.  physiolog. 
Chem.,  36  (1902). 

2  P.  A.  Levene  and  van  Slyke:  Biochem.  Zeitschr.,  13  (1908);  16 
(1909). 

3  H.  Malfatti:    Zeitschr.  f.  physiolog.  Chem.,  31  (1900). 

4  P.  Bergmann:    Skandinav.  Arch.  f.  Physiolog.,  18  (1906). 

5  Takamura:     Zeitschr.  f.  physiolog.  Chem.,  63  (1909). 

6  O.  Cohnheim:    Miinchener  med.  Wochenschr.,  1907. 


PROTEOLYTIC   ENZYMES  91 

erepsin,  does  not  dissolve  protein,  but  splits  peptones 
and  converts  them  into  amino-acids.  It  is  not  secreted, 
and  seems  to  be  absent  from  the  mucous  membrane  of 
the  cardiac  end  of  the  stomach.  It  is  found  in  extracts  of 
the  thick  mucous  membrane  of  the  antrum  pylori,  or  the 
pyloric  end.  One  might  think  that  we  were  dealing  with 
an  autolytic  enzyme,  or  tissue  enzyme  produced  for  the 
metabolism  of  the  tissue  (see  below).  But  the  protease 
or  the  erepsin  works  in  acid  solution  of  such  strength 
as  prevails  in  the  contents  leaving  the  pylorus,  and  it  is 
rather  probable  it  attacks  the  peptones  formed  by  the 
pepsin,  and  is  absorbed  in  the  stomach.  Moritz,  v. 
Mering,  and  others  had  observed  that  pure  water  or 
dilute  solutions  of  sugar  and  salts  run  rapidly  through  the 
stomach  without  being  absorbed  in  this  way.  But  I  was 
able  to  demonstrate  *  that  such  fluids  run  along  a  special 
channel  from  the  cardiac  end  to  the  pylorus — a  channel  to 
which  Waldeyer  has  given  the  name  "  magen-strasse." 
They  are  not  comparable  with  the  real  chyme  formed  in 
the  stomach-  by  the  digestion  of  food.  As  Tobler  2  has 
pointed  out,  by  means  of  a  really  natural  method,  thirty 
per  cent  of  the  nitrogen  of  the  meat  disappears  in  the 
stomach  of  the  dog.  In  the  cardiac  end,  the  mucous 
membrane  is  almost  wholly  composed  of  glands,  and 
between  them  are  left  only  very  small  ridges  covered  with 
mucous  cells,  which  can  scarcely  absorb  large  quantities. 
The  chief  absorption  must  occur  in  the  antrum  pylori 

1  O.  Cohnheim:    Munchener  rated.  Woch.,  1907. 

2  L.  Tobler:    Zeitschr.  f.  physiolog.  Chem.,  45  (1905). 


92  ENZYMES 

with  its  glands  less  closely  packed,  and  the  hydrochloric 
peptones  are  split  into  amino-acids  by  the  protease,  before 
they  pass  into  the  mucous  membrane.  Under  pathological 
conditions,  if  the  epithelial  layer  is  injured,  the  protease 
can  be  found  in  the  contents  of  the  stomach.  The  pres- 
ence of  amino-acids  in  the  stomach  is  used  as  a  diag- 
nostic method  which  demonstrates  injuries  of  the  epithelial 
layer  by  ulcer  or  cancer. 

The  trypsin  dissolves  most  natural  proteins,  with  two 
exceptions.  First,  the  genuine  colloidal  albumins,  as 
serum  albumin  or  egg  albumin  are  scarcely  or  not  affected 
at  all  by  trypsin;  raw,  fluid  egg-white  can  run  rapidly 
through  the  stomach,  and  then  it  is  not  digested,  but  is 
absorbed  unchanged,  and  eliminated  with  the  urine. 
Secondly,  the  collagen  of  connective  tissue,  as  was  first 
noted  by  Kiihne  and  Ewald,1  is  not  dissolved  by  trypsin. 
This  exception  is  used  by  Adolf  Schmidt 2  who  gives  to  a 
patient  raw  meat;  collagen-fibres  are  left  untouched  by 
trypsin,  and  their  occurrence  in  the  feces,  which  can 
readily  be  observed  microscopically,  is  an  evidence  of  the 
failure  of  pepsin  action. 

As  for  the  other  proteins,  if  the  whole  alimentary  canal 
and  reflexes  are  healthy,  the  lack  of  one  enzyme  or  of  one 
digestive  juice  can  be  compensated  for;  the  chief  regula- 
tion is  apparently  the  rhythmic  segmentations  described  by 


1  Kiihne  and  Ewald:     Heidelberger  Natur.   rrud.   Verein,  N.  F.,  i. 
(1876).— A.  Ewald:    Zeitschr.  f.  Biol.,  26  (1890). 

2  Adolf  Schmidt:     "Probekost,"   Wiesbaden,    1904. — R.   Baumstark 
and  O.  Cohnheim:    Zeitschr.  f.  physiol.  Chem.,  65  (1910). 


PROTEOLYTIC   ENZYMES  93 

Cannon,  by  which  the  small  intestine  squeezes  out  the 
chyme  until  all  digestible  substances  are  brought  into 
solution  and  absorbed.  The  capacity  of  compensation 
assures  the  safety  of  the  organism,  but  it  makes  physiologi- 
cal investigations  difficult.  We  can  deprive  a  dog  of  the 
whole  stomach,  and  yet  the  animal  can  digest  very  well; 
apparently  the  stomach  is  not  necessary  for  life.  If  we 
now  extirpate  the  pancreas,  the  dog  will  die.  On  the 
contrary,  when  we  deprive  a  dog  of  the  pancreas,  we 
cause  but  few  disturbances  of  digestion;  the  pancreas  is 
not  necessary  for  life.  When  we  now  extirpate  the 
stomach,  however,  the  dog  dies. 

Proteins  dissolved  by  trypsin  are  converted  first  into 
peptones,  which,  however,  clearly  differ  from  pepsin 
peptones.  Afterward,  they  are  converted  to  a  great 
extent  into  amino-acids.  Whether  all  amino-acids  must 
be  derived  from  peptones,  is  not  certain.  The  question 
involves  knowledge  of  the  configuration  of  the  protein 
molecule,  and  especially  of  the  different  linkings  of  the 
amino-acids,  and  I  cannot  discuss  this  fully.  We  may, 
however,  note  the  following  facts:  Immediately  after  the 
beginning  of  the  action  of  trypsin,  amino-acids  are  split 
off,  while  the  rest  of  the  protein  is  still  untouched.  This 
residue  gradually  becomes  smaller,  but  the  whole  mole- 
cule is  never  disintegrated.  The  remainder  contains 
phenylalanine,  proline,  glycocoll,  and  glutaminic  acid, 
while  tyrosine,  tryptophane,  and  cystine  are  set  free  com- 
pletely by  trypsin.  Leucine,  lysine,  arginine,  and  aspartic 
acid  are  partly  liberated.     Like  natural  proteins,  peptones 


94  ENZYMES 

formed  by  peptic  digestion  are  partly  dissociated  by 
trypsin;  they  give  rise  both  to  tyrosine  and  other  amino- 
acids,  and  to  peptones  which  vigorously  resist  further 
dissociation  by  means  of  trypsin.  The  remainder,  which 
is  not  acted  upon,  Kiihne  called  the  "antigroup."  We 
do  not  know  whether  the  amino-acids  are  linked  here  in  a 
different  manner,  or  what  the  reason  may  be.  The 
artificial  peptids,  di-  and  poly-peptids,  built  up  by  E. 
Fischer,  are  also  only  partly  dissociated  by  trypsin.  It 
is  impossible  now  to  foretell  whether  a  peptid  is  touched 
or  not,  or  to  tell  why  alanylglycine  is  dissociated,  and 
glycylglycine  not,  why  tetraglycylglycine  is  dissociated,  and 
triglycylglycine  not,  etc.  Physiologically,  the  differences 
have  no  weight,  because  erepsin  dissociates  all  peptones 
and  all  artificial  peptids.  Thus  the  peptones  formed  by 
trypsin  are  always  intermediate  compounds,  and  disappear 
after  a  short  time.  For  the  chemistry  of  enzymes  and  of 
proteins,  these  observations  may  become  very  important. 
At  present  they  are  isolated  facts,  which  do  not  lend  them- 
selves to  explanation. 

Trypsin  is  secreted  as  zymogen,  and  is  completed  or 
activated  by  enterokinase.  This  holds  good,  however, 
only  for  the  pancreatic  juice  secreted  after  the  stimulus 
by  secretin.  The  pancreatic  juice  secreted  after  nervous 
stimulus,  and  after  the  stimulation  of  the  intestine  by 
fats  or  soaps,  contains,  besides  trypsinogen,  ready  efficient 
trypsin.  The  juice  secreted  by  meat  is  an  almost  pure 
secretin-juice,  and  contains  no  trypsin  or  but  very  little, 
but  dogs  eating  a  mixed  food  secrete  a  juice  which  con- 


PROTEOLYTIC   ENZYMES  95 

tains  both.  Discrepancies  among  authors  are  apparently 
due  to  these  differences  in  the  secretion  under  different 
conditions,  but  these  differences  have  led  to  an  observation 
of  very  great  importance.  Accordingly  as  dogs  are  fed 
on  a  meat  diet  or  a  meatless  diet,  they  secrete  different 
pancreatic  juices,  and  if  we  suddenly  change  the  diet,  the 
juice  is  not  changed  suddenly,  but  only  gradually  during 
many  days,  so  that  during  the  interval  digestion  may  be 
disturbed.  So  far  as  I  can  see,  it  is  the  first  experimental 
observation  which  explains  the  great  influence  of  alteration 
of  diet  on  man. 

For  other  adaptations  there  is  no  evidence.  Like 
ptyalin — see  above — the  amount  of  trypsin  seems  to 
differ  in  juices  provoked  by  different  stimuli.  But  a 
special  adaptation  of  trypsin  in  case  of  need,  according 
to  the  amount  of  proteins  or  the  digestibility  of  proteins, 
has  not  been  demonstrated.1 

Some  authors  have  thought  that  trypsin  is  not  one 
enzyme,  but  a  group  of  proteolytic  enzymes,  one  of  which 
attacks  casein,  one  gelatin,  one  fibrin,  etc.  I  do  not  think 
that  the  experiments  2  which  are  cited  are  conclusive.  If 
a  weak  trypsin  solution  digests  casein  at  370  C,  and  does 
not  digest  gelatin  at  230  C,  the  difference  does  not  allow 
the  suggestion  of  two  enzymes. 


1  J.  P.  Pawlow:    Nagel's  "Handbuch  d.  Physiologie,"  ii. 

2  O.  Cohnheim:  Zeitschr.  f.  physiolog.  Chem.,  33  (1901);  35,  36 
(1902);  47,  49  (1906);  51,  52  (1907).— S.  S.  Salaskin:  ibid.,  35  (1902).— 
F.  Kutscher  and  J.  Seeman:  ibid.,  35  (1902). — J.  H.  Hamburger  and  J. 
Hekma:    Akad.  v.  Wetenschappen,  Amsterdam,  1902. 


96  ENZYMES 

Like  other  proteolytic  enzymes,  trypsin  provokes  curd* 
ling  of  milk,  and  dissolves  again  the  clotted  casein.  Because 
efficient  trypsin  redissolves  casein  very  rapidly,  pancreatic 
rennin  has  been  less  studied  than  gastric  rennin. 

The  last  proteolytic  enzymes  to  be  mentioned  are  those 
of  the  small  intestine, — erepsin  and  arginase. 

Erepsin  is  found  in  the  enteric  juice,  and  can  always 
be  extracted  in  great  amount  from  the  intestinal  mucous 
membrane  ground  with  sand.  It  disintegrates  all  pep- 
tones and  converts  them  into  amino-acids,  and  it  also 
disintegrates  all  artificial  peptids.1  In  experiments  on  the 
efficiency  of  ereptic  dissociation,  it  is  necessary  to  work 
with  the  compounds  which  are  naturally  acted  upon  by 
erepsin.  Proteoses  are  converted  by  erepsin;  but  it  is 
quite  well  known  that  they  reach  the  intestine  in  but  very 
small  amount.  The  designation  "peptone,"  applied  to 
many  purchaseable  products  which  yield  chiefly  pro- 
teoses, has  resulted  in  much  error.  The  peptones  formed 
by  natural  peptic  digestion  are  rapidly  converted  by 
erepsin.  The  mass  of  meat-peptones  that  leaves  the 
dog's  stomach  in  three  hours,  is  split  by  the  extract  of  a 
dog's  intestine  in  two  hours.  The  extract  of  the  intestines 
of  two  dogs  completely  converts  in  three  hours  30  gm.  of 
peptones  formed  in  peptic  digestion.  One-fourth  of  the 
extract  of  one  intestine  converts  7  gm.  in  two  hours.  In 
enzyme-investigations,  many  authors  have  forgotten  that 
a  man  metabolizes  100  gm.  of  protein  per  day,  and  that 

1  E.  Abderhalden  and  Y.  Teruuchi:  Zeitschr.  f.  physiolog.  Chem., 
49  (1906). 


PROTEOLYTIC   ENZYMES  97 

an  enzyme  which  digests  i  gm.  of  fibrin  in  12  hours,  or 
the  proteins  of  a  small  organ  in  four  months,  cannot  be  a 
true  digestive  enzyme,  as  erepsin  is.  Among  the  natural 
complex  proteins,  protamine  is  split  by  erepsin,  histone 
and  casein  are  slowly  and  partly  converted,  and  all  other 
proteins  are  left  untouched.  That  histones  are  attacked 
by  erepsin,  may  be  of  some  importance,  because  most 
probably  all  tissues  yield  histones,  and  the  so-called  auto- 
lytic  enzymes  resemble  erepsin  perhaps  more  than  trypsin. 

Erepsin  is  found  in  the  human  fetus  in  the  seventh 
month.1     It  can  also  be  found  in  the  feces.2 

Arginase,  found  in  the  liver  and  intestine  by  Kossel  and 
Dakin,  splits  arginine  into  ornithine  and  urea: 
NH  0 


NH2.C 

NH2.C.NH2 

NH 

and 

H.C.H 

+ 

H20  = 

H2CNH2 

H.C.H 

HCH 

H.C.H 

HCH 

H.C.NH2 

HCNH2 

COOH 

COOH 

1 E.  Jaeggy:  Zentralbl.  f.  Gynak.,  1907. — L.  Langstein  and  M. 
Soldin,  Jahrbuch  f.  Kinderheilkunde,  67  (1908). — D.  E.  Edsall:  Journ. 
Amer.  Med.  Association,  1907. 

2  A.  Schittenhelm  and  Fr.  Frank:  Zeitschr.  f.  experim.  Path.  u. 
Therap.,  8  (1910). 

7 


98  ENZYMES 

It  completes  the  action  of  erepsin,  loosening  the  second 
linking  of  amino-acids  in  the  protein-molecule,  besides 
the  peptid  linking. 

The  occurrence  of  arginase  in  the  enteric  juice  has  not 
been  investigated,  but  the  extract  contains  it  in  much 
smaller  amount  than  the  liver  extract,  so  that  it  is  not 
certain  whether  we  have  in  arginase  a  digestive  or  a  so- 
called  autolytic  enzyme.  Perhaps  arginase  has  a  function 
in  the  particular  metabolism  of  the  intestine  and  liver, 
perhaps,  like  the  peptolytic  enzyme  of  the  stomach,  it  may 
attack  arginine  in  its  course  of  absorption.  So  long  as  we 
do  not  know  the  fate  of  the  absorbed  protein  or  the  inter- 
mediate metabolism  of  proteins,  the  question  cannot  be 
answered  with  certainty. 


CHAPTER  XIV 

Miscellaneous  and  Vegetable  Enzymes 

Besides  carbohydrates,  fats,  and  proteins,  food  contains 
other  compounds,  such  as  lecithin,  nucleic  acid,  and 
cholesterin,  which  are  disintegrated  in  the  alimentary 
canal. 

Nucleic  acid  becomes  insoluble  in  the  stomach,1  because 
it  forms  salts  with  proteins  and  proteoses  which  are 
insoluble  in  an  acid  medium.  This  precipitate  was  called 
"nuclein"  by  earlier  authors,  and  the  undissolved  remnant 
of  peptic  digestions  of  meat  or  tissues  is  mainly  this 
nuclein.  This  remnant  is  gradually  dissolved,  and  the 
nucleic  acid  is  absorbed  in  the  small  intestine.2  It  is 
possible  that  there  is  a  simple  solution  of  nucleic  acid  in 
the  alkaline  pancreatic  juice,  and  that  portions  of  the 
nucleic  acid  can  pass  the  epithelial  cell  and  enter  the 
blood  without  dissociating.  Probably  it  is  converted  in 
the  lumen  of  the  intestine,  or  in  the  course  of  absorption. 
Kutscher 3  allowed  pancreas  and  mixtures  of  pancreas 

1  F.  Umber:    Zeitschr.  f.  klin.  Med.,  43  (1901). 

2  Gumlich:  Zeitschr.  f.  physiolog.  Chem.,  18  (1893). — T.  H.  Milroy: 
ibid.,  22  (1896). — P.  M.  Popoff:  ibid.,  18  (1893). — E.  Abderhalden  and 
A.  Schittenhelm:  ibid.,  48  (1906). — T.  Araki:  ibid.,  38  (1903). — F. 
Sachs:    ibid.,  46  (1905). — M.  Nakayama:    ibid.,  41  (1904). 

3  F.  Kutscher:  Zeitschr.  f.  physiolog.  Chem.,  32  (1900);  39  (1903);  44 
(i9°5)- 

99 


100  ENZYMES 

and  intestine  to  stand  for  a  long  time  at  the  temperature 
of  the  body.  As  a  result,  he  found,  besides  the  cleavage 
products  of  proteins,  purine  bases  and  pyrimidines,  which 
are  the  well-known  dissociation  products  of  nucleic  acid. 
Therefore,  pancreas  and  intestine  must  contain  nuclease, 
and  Sachs,  Nakayama,  and  others  have  made  extracts  of 
the  gland  and  the  mucous  membrane,  and  have  studied 
their  action  on  nucleic  acid  or  nucleic  salts.  The  nucleic 
acid  was  converted  into  the  co-called  nucleic  acid-fr,  a 
compound  of  different  physical  properties;  the  further 
conversion  into  purine  bases,  pyrimidines,  and  phosphoric 
acid  could  be  observed  only  in  small  amount,  because  nu- 
clease loses  its  activity  very  rapidly  in  solutions  containing 
trypsin  or  other  proteolytic  enzymes.  But  I  think  that 
conversion  would  proceed  more  readily  under  natural 
conditions,  and  that  nucleic  acid  is  split  to  a  great  extent 
during  absorption. 

The  precipitation  of  nucleic  acid  in  the  stomach  and  its 
solution  by  pancreatic  nuclease  have  been  used  by  Adolf 
Schmidt  as  a  clinical  method.  Schmidt  observed  that, 
under  normal  conditions,  nuclei,  for  instance  of  the  calf's 
thymus,  disappear  in  the  intestine,  but  he  was  able  to 
find  nuclei  in  the  feces  of  pathological  cases.  He  made 
the  inference  that  in  these  cases  pancreatic  secretion  is 
checked.  If  he  is  right,  intestinal  nuclease  would  be  no 
digestive  enzyme.  The  occurrence  of  nuclease  in  all 
tissues  prevents  us  here  from  drawing  a  clear  conclusion. 

Lecithin  resembles  fats  in  chemical  configuration  and 
physical  behavior,  solubility,  and  most  properties.     Like 


MISCELLANEOUS   AND   VEGETABLE   ENZYMES        101 

fats,  it  consists  of  glycerin-esters  of  fatty  acids.  Lecithin 
differs  from  fat  in  the  further  combination  of  glycerin  with 
phosphoric  acid  and  the  base  choline.  It  is  an  essential 
part  of  every  living  cell,  therefore  food  nearly  always 
contains  lecithin.  Kutscher  and  Lohmann,1  and  Bergell, 
demonstrated  that  the  pancreas  and  the  intestine  yield  an 
enzyme  which  disintegrates  lecithin.  They  allowed  pan- 
creas and  intestine  to  stand  for  several  months  and  digest 
its  own  substance,  and  they  then  found  choline  in  large 
quantities.  We  have  no  further  evidence  for  the  secre- 
tion of  this  enzyme,  but  it  is  probable  that  lecithin  is 
digested  in  the  intestine,  and  it  is  also  probable  that 
lipases  dissociating  other  esters  attack  lecithin  as  well.2 

Animal  food  often  contains  haemoglobin  and  its  non-pro- 
tein constituent,  haematin.  Haemoglobin  is  disintegrated 
by  the  acid  of  gastric  juice,3  and  haematin  must  be  present 
everywhere  in  the  intestine,  because  it  contains  iron  in 
non-ionic  form,  and,  according  to  the  microscopical 
studies  of  Quincke  and  Hochbauer,4  iron  can  be  found 
in  ionic  form  in  the  absorbing  cells  of  the  duodenum  and 
in  the  upper  small  intestine.     The  enzyme  is  unknown. 

Cholesterin  seems  to  be  transformed  only  by  bacteria, 


1  F.  Kutscher  and  Lohmann:  Zeitschr.  f.  physiolog.  Chem.,  39  (1903); 
41  (1904). 

2  C.  Schumoff-Simanowski  and  N.  Sieber:  Zeitschr.  f.  physiolog. 
Chem.,  49  (1906). 

3R.  v.  Zeynek:    ibid.,  30  (1899). 

4H.  Hochbauer  and  H.  Quincke:  Arch.  f.  exper.  Path.  u.  Pharmak., 
37  (1896). — E.  Abderhalden:  Zeitschr.  f.  Biol.,  39  (1900). — A.  Hofmann: 
Virchow's  Arch.,  151  (1898). 


102  ENZYMES 

and  for  many  years  it  has  been  the  general  opinion  that 
the  cellulose  of  vegetable  food  is  also  dissolved  and  dissoci- 
ated by  the  bacteria  of  the  alimentary  canal.  No  one  has 
observed  any  influence  of  any  digestive  juice  or  extract  of 
digestive  organs  upon  cellulose,  and  this  indigestibility 
is  a  matter  of  great  biological  importance.  Nevertheless, 
cellulose  disappears  in  passing  through  the  alimentary 
canal  of  herbivorous  animals,  and  even  in  man,  some  is 
digested.1  The  action  of  micro-organisms  takes  place 
in  the  large  intestine,  and  in  the  ruminants  in  the  first 
stomach.  This  action  has  been  studied  in  vitro  by  Tap- 
peiner.2  In  human  pathology,  it  was  found  by  Adolf 
Schmidt3  that  the  amount  of  cellulose  which  disappears 
in  passing  through  the  alimentary  canal  of  man  varies 
greatly,  but  without  any  evidence  for  differences  in  the 
quantity  or  kind  of  bacteria.  It  is  not  impossible  that 
for  the  disintegration  of  cellulose  a  co-operation  of  bacteria 
and  the  living  cell  wall  of  the  intestine  is  necessary.  For 
the  part  played  by  the  living  wall  I  have  three  examples : 

i.  As  Schutz  4  has  pointed  out,  if  we  introduce  a  great 
quantity  of  bacteria,  such  as  Bacillus  pyocyaneus,  through 
a  fistula  into  the  small  intestine  of  a  dog  or  cat,  the  bacteria 
are  killed  in  a  very  short  time.  The  surviving  intestine 
removed  from  the  body  and  kept  in  sterilized  blood,  acts 


1  W.  v.  Knieriem:    Zeitschr.  f.  Biol.,  21  (1885). 

2  H.  Tappeiner:    ibid.,  19,  20  (1884). 

3H.  Lohrisch:     Zeitschr.  f.  physiolog.  Chem.,  47  (1906). 
*  R.  Schutz:     Arch.  f.  Verdauungskrankheiten,  7  (1901). — Verh.  des 
Kongresses  f.  innere  Medizin,  1909. 


MISCELLANEOUS   AND    VEGETABLE   ENZYMES         103 

similarly.  But  the  surviving  organ  loses  its  power  thirty 
minutes  after  removal  from  the  body,  at  the  same  time 
that  the  epithelial  cell  loses  its  absorbing  power.  Neither 
the  dead  nor  the  dying  cells,  nor  extracts  of  the  mucous 
membrane,  nor  any  secretion,  can  kill  bacteria,  but  only 
the  living  cell  with  its  structures  can  do  so. 

2.  A  lower  marine  invertebrate,  Actinia,1  the  well-known 
sea  anemone,  has  often  been  investigated,  because  it  can 
digest  great  quantities  of  meat,  fibrin,  or  other  proteins 
without  secreting  any  digestive  fluid.  According  to  the 
observations  of  many  authors,  Chapeavese,  Mesnil,  and 
others,  the  mesenteric  filaments,  the  digestive  organs  of 
these  animals,  can  dissolve  and  dissociate  protein,  but 
only  when  they  are  in  immediate  contact  with  it. 

3.  The  surviving  wall  of  the  intestine  converts  pep- 
tones in  another  way  than  do  the  extracts  of  the  mucous 
membrane. 

It  is  possible  that,  in  the  stomach  of  living  ruminants, 
and  in  the  cecum  and  the  large  intestine  of  man  and 
herbivora,  bacteria  find  conditions  so  much  better  than 
we  can  imitate,  that  they  dissolve  cellulose  in  much  greater 
quantity  than  in  Tappeiner's  experiments.  But  it  is  also 
possible  that  they  carry  on  the  digestion  of  cellulose  in  a 
manner  unknown  at  the  present  time. 

Through  the  whole  series  of  invertebrates  we  find  no 
difference  in  the  occurrence  and  arrangement  of  the 
digestive   enzymes,   except   in  the   cases  of  lactase  and 

1  O.  and  R.  Herting:     "Die  Aktinien,"  Jena,  1879. 


104  ENZYMES 

salivary  ptyalin,  already  mentioned.  The  pancreatic 
ferments,  gastric  pepsin  and  intestinal  erepsin  and  invertin, 
seem  to  be  identical  in  mammals,  birds,  frogs,  and  fish. 
And  so  does  the  association  between  the  intestine  and 
pancreas,  or  intestine  and  stomach.  The  digestive 
organs  of  invertebrates  show  many  differences  in  arrange- 
ment and  development,  according  to  the  biological  need 
of  the  species.  But  their  tools  are  the  same  enzymes 
described  above  in  mammals,  namely,  ptyalin,  maltase, 
invertin,  steapsin,  and  nuclease.  Only,  instead  of  the 
three  proteolytic  enzymes  adapted  to  one  another,  we  find 
one  enzyme  which  dissolves  proteins  and,  at  the  same 
time,  converts  them,  partially  or  completely,  into  amino- 
acids  as  thoroughly  as  do  trypsin  and  erepsin.  Most  of 
these  enzymes  work  in  a  neutral  reaction,  some  are 
assisted  by  an  acid  reaction,  like  pepsin,  and  split  proteins 
completely.1  In  one  of  the  large  higher  invertebrates,  the 
Octopus  vulgaris,  a  cephalopod,  it  was  possible  to  show 
that  the  secretion  of  enzyme  was  governed  by  the  nervous 
system.2  In  actinia  and  some  other  lower  invertebrates, 
it  seems  that  enzymes  act  only  in  vacuoles  of  the  proto- 
plasm, and  are  not  excreted  from  the  cells.  In  these  cases 
digestible  substances  are  surrounded  by  moving  proto- 
plasm and  digested  in  the  space  thus  formed.  In  protozoa 
this  kind  of  digestion  has  been  observed  by  Nierenstein,3 

1  L.  B.  Mendel  and  H.  C.  Bradley:     Amer.  Journ.  of  Physiol.,   13 

(1905)- 

2  O.  Cohnheim:    Zeitschr.  f.  physiolog.  Chem.,  35  (1902). 

3  E.  Nierenstein:    Zeitschr.  f.  allgem.  Physiolog.,  5  (1905). 


MISCELLANEOUS   AND   VEGETABLE   ENZYMES        105 

who  saw  that  digestible  substances  were  brought  into  a 
vacuole,  that  acid  and  enzyme  were  secreted  into  the 
vacuole,  and  that  proteins  were  thus  dissolved.  Digestion 
occurs  inside  of  the  cell,  but  outside  of  the  protoplasm. 

Bacteria  yield  all  types  of  enzymes:  sucroclastic 
enzymes,  lipases  or  steapsins,  nucleases,1  lecithases,  and 
proteolytic  enzymes.  In  some  cases,  it  has  been  possible 
to  extract  the  enzymes  from  the  bacteria  and  investigate 
them  in  solution.  In  most  cases  only  the  action  of  the 
bacteria  themselves  upon  proteins  or  sugars  has  been 
observed,  and  conclusions  have  been  drawn  as  to  the 
existence  of  corresponding  ferments  in  the  bacteria.  The 
proteolytic  enzymes  of  yeast 2  and  other  micro-organisms 
closely  resemble  trypsin,  and  have  received  the  name 
"endotrypsin."  They  give  rise  to  the  ordinary  amino- 
acids,  but  in  such  experiments  the  amino-acids  are  often 
converted  into  acids,  keto-acids,  alcohols,  etc.,  because 
in  these  unicellular  organisms  digestive  enzymes  and 
metabolism-enzymes  cannot  be  separated  (see  below). 

Among  plants,  full-grown  individuals  have  no  special 
digestive  organs  like  animals,  but  in  very  young  plants  the 
germ  is  surrounded  by  storage-substances  which  are 
used  for  food  in  growth;  and  these  substances,  starch, 
fat,  and  proteins,  are  digested  or  dissolved  by  enzymes 
secreted  by  the  growing  germ.     Therefore  germinating 

1  F.  Kutscher:    Zeitschr.  f.  physiolog.  Chem.,  32  (1900);  39  (1903). 

2  M.  Hahn  and  Geret:  Zeitschr.  f.  Biol.,  40  (1900). — F.  Kutscher: 
Zeitschr.  f.  physiolog.  Chem.,  32  (1900);  34  (1902). — E.  Salkowski: 
ibid.,  13  (1889). 


106  ENZYMES 

seeds  yield  powerful  enzymes,  which  act  outside  the 
protoplasm,  and  can  easily  be  obtained  and  purified.  The 
diastase  of  seeds  is  one  of  the  longest  known  enzymes: 
the  steapsins  of  the  seeds  of  the  castor-oil  plant  are  used 
in  the  manufacture  of  soaps  from  oils.1  I  have  mentioned 
that  this  steapsin  acts  well  only  in  an  acid  medium,  and 
that  this  acid  is  produced  by  converting  the  carbohydrates 
of  the  seed  into  organic  acid  at  the  time  of  germination. 
The  proteolytic  enzymes  of  germinating  seeds  have  been 
thoroughly  studied  by  E.  Schulze 2  in  Zurich.  Under 
normal  conditions,  the  cleavage  products  formed  by  these 
enzymes  are  used  for  building  up  new  proteins,  but 
Schulze  could  check  the  synthetic  power  of  germinating 
plants  by  keeping  the  latter  in  dark  rooms.  In  such 
"etiolated"  plants,  the  proteolytic  enzyme  gives  rise  to 
the  ordinary  amino-acids  just  as  do  strong  boiling  acids 
or  trypsin  and  erepsin.  Furthermore,  some  fruits  which 
resemble  seeds  from  a  biological  point  of  view,  yield 
powerful  proteolytic  enzymes,  e.g.,  the  bromelin  of  the 
pineapple  3  and  the  papain  or  papayotin  of  the  papaw 
plant,4  which  have  often  been  studied.  They  give  rise 
both  to  peptones  and  amino-acids,  and  we  cannot  tell 
with  certainty  whether  they  consist  of  two  enzymes,  i.e., 

1 W.   Connstein:     Ergebnisse  d.   Physiologie;   iii.,   Biochemie,    1904. 

2  E.  Schulze:  Zeitschr.  f.  physiolog.  Chem.,  24  (1898);  26  (1899);  30 
(1900);   47  (1906). 

3  R.  H.  Chittenden:    Journ.  of  Physiol.,  15  (1883). 

4  R.  Neumeister:  Zeitschr.  f.  Biol.,  26  (1890). — L.  B.  Mendel,  Con- 
necticut Academy,  1901;  Amer.  Journ.  of  Medical  Sciences,  1902. — 
F.  Kutscher:    Zeitschr.  f.  phys.  Chem.,  46  (1905). 


MISCELLANEOUS   AND    VEGETABLE   ENZYMES         107 

a  zymogen  and  activator  like  pepsin  and  erepsin,  as  has 
been  suggested  by  Vines,1  or  of  one  enzyme,  like  trypsin, 
which  splits  proteins  partially.  Nor  do  we  know  if  in 
plants  germinating  proteins  are  and  must  be  completely 
disintegrated  as  in  animal  digestion.  Proteolytic  enzymes 
are  found  in  flowers,  leaves,  and  other  parts  of  plants,2 
but  they  occur  in  smaller  amount  or  are  less  powerful  than 
the  enzymes  of  seeds.  They  do  not  resemble  the  digestive 
enzymes,  but  the  autolytic  enzymes  of  tissues. 


1  S.  H.  Vines:    Annals  of  Botany,  17-20  (1903-1906). 
3  A.  Kossel:    Zeitschr.  f.  physiolog.  Chem.,  49  (1906). 


CHAPTER  XV 

The  Hydrolytic  Enzymes  of  Tissues,  or  Auto- 
lytic  Enzymes 

For  reasons  mentioned,  the  knowledge  of  these  enzymes, 
which  are  not  secreted  and  not  destined  for  secretion,  is 
rather  incomplete.  These  enzymes  have  been  studied  par- 
ticularly in  recent  years,  first  because  we  have  gradually 
learned  better  methods  of  obtaining  them,  and  secondly, 
on  account  of  their  supposed  biological  and  clinical  in- 
terest. We  know,  since  the  labors  of  Voit,  that  the  non- 
infectious diseases,  like  diabetes  and  gout,  do  not  affect 
the  general  metabolism  of  our  body.  The  quantity  and 
character  of  the  products  which  are  finally  formed  in  the 
metabolism  of  proteins  or  carbohydrates,  is  the  same  in 
disease  as  in  health.  Therefore,  many  physiologists  and 
physicians  have  hoped  that  the  intermediate  metabolism 
intercalated  between  the  digestion  organs  and  the  organs 
of  elimination,  could  give  us  some  explanation  of  the 
riddles  of  disease.  In  recent  years  much  has  been  done 
regarding  the  enzymotic  processes  taking  place  in  tissues, 
and  the  enzymes  responsible  for  these  processes. 

We  can  separate  the  tissue-enzymes  into  three  classes: 

first,  the  hydrolytic;   second,  the  oxidizing;  and  third,  the 

metabolism  enzymes. 

The  hydrolytic  enzymes  of  tissues  are  mainly  the  same 

1 08 


THE   HYDROLYTIC   ENZYMES   OF   TISSUES  109 

as  the  enzymes  of  the  alimentary  canal.  We  have  no 
evidence  of  any  difference  in  properties,  solubility,  or 
action,  between  the  diastase  in  saliva  or  pancreatic  juice, 
and  the  diastase  in  the  liver,  the  muscles,  or  the  blood; 
or  between  the  lipase  of  the  pancreatic  juice  and  that  of 
the  liver.  The  only  difference  has  been  mentioned  before, 
i.e.,  the  difficulty  of  extracting  the  intracellular  enzymes, 
and  on  account  of  this  difficulty  we  may  be  deceived  by 
delusive  differences  obtained  in  their  use. 

Another  difficulty  is  the  separation  of  blood-enzymes 
from  the  tissue-enzymes,  i.e.,  the  removal  of  blood  from 
organs.  Washing  out  blood  completely  is  impossible 
post  mortem,  and  is  not  easy  during  life.1 

1  O.  Cohnheim  and  D.  Pletnew:  Zeitschr.  f.  physiolog.  Chem.,  68 
(1910). 


CHAPTER  XVI 

Proteolytic  Enzymes  of  Blood 

Under  normal  conditions,  blood  plasma  does  not  con- 
tain enzymes;  but  the  blood  plasma  is  the  path  for  all 
substances  from  one  organ  to  another,  from  the  places  of 
absorption  to  the  places  of  utilization  and  elimination. 
It  is  possible  that  the  enzymes  of  the  alimentary  canal, 
which  are  found  in  the  products  of  their  action,  are 
absorbed  together  with  these  products.  Pepsin  is  found 
in  small  amount  in  the  urine,1  and  before  elimination, 
pepsin  must  have  passed  through  the  blood.  Weinland  2 
has  studied  this  question  by  means  of  invertin,  which 
occurs  only  in  the  mucous  membrane  of  the  small  intestine, 
and  nowhere  else  in  the  bodv,  and  of  which  the  smallest 
traces  can  be  recognized.  Blood  plasma  contains  no 
invertin,  but  it  was  possible  to  find  the  invertin  in  the 
plasma  after  repeated  subcutaneous  injections  of  cane 
sugar  in  puppies.  The  experiment  gives  evidence  that 
cells  producing  enzymes  and  secreting  them  in  one  direc- 
tion, can  change  this  one-sided  polarity  and  throw  the 
enzyme  backward  into  the  blood,  when  the  stimulus 
comes  to  them  from  the  opposite  direction.  But  we  learn 
further  from  the  experiment  that,  under  normal  conditions, 
cells  deliver  the  enzymes  in  only  one  direction  and  that 

1  M.  Matthes:    Arch.  f.  exper.  Path.  u.  Phar.,  40  (1904). 

2  E.  Weinland:    Zeitschr.  f.  Biol.,  47  (1905). 

no 


PROTEOLYTIC   ENZYMES    OF    BLOOD  111 

blood-plasma  contains  no  enzymes  arising  from  the 
digestive  organs.  Only  when  the  cells  are  destroyed,  as 
in  phosphorus  poisoning,  do  they,  and  with  them  the 
enzymes,  enter  and  circulate  in  the  blood  in  great  amount.1 
As  blood  clots,  fibrin  is  formed,  but  the  clots  are  dissolved 
in  a  short  time.  The  livers  of  dogs  or  men  killed  by 
phosphorus  soften  and  dissolve  after  a  few  hours. 

All  blood  corpuscles  contain  enzymes.  In  red  cor- 
puscles 2  and  in  blood-platelets,  proteolytic  enzymes  are 
found,  which  convert  peptids  into  amino-acids  like 
erepsin.  This  ferment  of  blood-platelets  is,  according  to 
Deetjen,  involved  in  the  clotting  of  blood  (see  below). 
More  important  are  the  proteolytic  enzymes  of  the  white 
corpuscles.  The  white  corpuscles  are  complete  organisms, 
independent  in  many  respects  of  the  rest  of  the  body,  and 
living  like  protozoa.  They  eat  food,  take  in  oxygen  and 
give  out  carbonic  acid,  dissolve  and  dissociate  solid 
proteins  such  as  fibrin,  digest  the  bodies  of  micro-organisms 
and  store  up  glycogen,  fat,  and  other  "granula."  Leu- 
cocytes occasion  the  resolution  of  the  solid  exudate  in  the 
lungs  of  croupous  pneumonia,  and  develop  the  proteolytic 
power  of  pus  which  softens  the  tissues.  That  white 
corpuscles  contain  a  proteolytic  enzyme,  has  been  known 
for  a  long  time.     Recently,  Opie  3  has  pointed  out  that 


1  M.  Jacoby:    Zeitschr.  f.  physiolog.  Chem.,  30  (1900). — A.  J.  Wake- 
man:    ibid.,  44  (1905). 

2  E,  Abderhalden  and  H.  Deetjen:    ibid.,  51  and  53  (1907). 

3  E.  L.   Opie:     "Studies  of  the  Rockefeller  Institute,"  4  (1905);  6 
(1906);   8  (1908). — A.  R.  Dochez:     10  (1910). 


112  ENZYMES 

two  different  enzymes  occur  in  those  leucocytes  which  act 
especially  as  phagocytes.  The  polynuclear  leucocytes 
with  fine  granulations  contain  an  enzyme  which  causes 
proteolytic  digestion  in  a  neutral  or  alkaline  medium,  and 
which  is  almost  wholly  incapable  of  action  when  placed 
in  an  acid  medium.  Opie  gives  to  this  enzyme  the  name 
"  leucoprotease."  The  large  mononuclear  leucocytes,  the 
macrophages  of  Metchnikoff,  contain  an  enzyme  which 
is  incapable  of  digesting  protein  in  an  alkaline  medium, 
but  is  active  in  the  presence  of  a  weak  acid,  for  instance 
0.2  per  cent  acetic  acid.  Stronger  acid,  or  hydrochloric 
acid,  checks  the  action.  Opie  suggests  for  this  enzyme  the 
name  "  lymphoprotease." 

In  life,  both  enzymes  are  limited  to  intracellular  action. 
It  is  known  that  intracellular  digestion  by  many  unicellu- 
lar organisms,  amoeba  and  others,  occurs  in  the  presence 
of  acid.  The  protoplasm  manufactures  a  little  temporary 
stomach  within  its  substance.  Similarly,  leucocytes  en- 
gulf and  ingest  bacteria  and  other  micro-organisms,  red 
corpuscles,  etc.,  and  by  means  of  their  enzymes  they 
dissolve  the  protein  of  these  structures.  These  enzymes 
are  not  involved  in  the  digestion,  nutrition,  or  metabolism 
of  the  human  or  animal  body,  but  in  phagocytosis  and 
immunity. 

The  conditions  are  changed  after  the  death  and  mechani- 
cal or-  chemical  disintegration  of  leucocytes.  Then,  leuco- 
and  lymphoprotease  are  set  free,  and  can  either  attack 
the  proteins  of  tissues  outside  of  the  leucocytes,  or  the  ac- 
tion is  checked  by  the  anti-enzymotic  action  of  the  serum 


PROTEOLYTIC   ENZYMES    OF   BLOOD  113 

albumin  surrounding  cells  (vide  supra).  Investigation 
of  the  proteolytic  enzymes  of  leucocytes  is  difficult.  We 
meet  with  two  different  enzymes  having  different  op- 
tima and  different  degrees  of  resistance  to  high  tempera- 
ture, and  we  meet  with  a  substance  which  can  check  the 
action.  The  detection  of  these  enzymes  is  now  facilitated 
by  using  a  plate  of  coagulated  blood  according  to  the 
method  of  Miiller  and  Jochmann.1  Traces  of  enzyme 
brought  on  the  surface  of  the  plate  are  detected  by  the  ap- 
pearance of  a  cavity  formed  by  solution  of  a  portion  of 
the  solid  protein  of  the  plate. 

There  can  be  no  doubt  that  the  traces  of  proteolytic 
enzymes  occasionally  found  in  serum  or  in  fibrin,2  are 
the  enzymes  derived  from  dissolved  leucocytes.  The 
enzymes  of  inflammation  and  pus,  which  destroy  tissues, 
are  leuco-  and  lymphoprotease.  Opie  suggests — and  I 
think  he  is  right — that  the  enzyme  observed  by  him  in 
bone  marrow  is  identical  with  leucoprotease,  and  the 
enzyme  of  lymphatic  gland  with  lymphoprotease.  From 
the  spleen,  Hedin 3  has  extracted  two  enzymes,  one  acting 
in  an  acid,  the  other  in  an  alkaline  medium,  and  to  which 
he  gives  the  names  lienoprotease  a  and  /?.  The  great 
quantity  of  all  forms  of  leucocytes  in  the  spleen  suggests 
to  us  the  identity  of  these  with  Opie's  enzymes.     Disinte- 

1  E.  Miiller  and  Jochmann:  Miinchener  med.  Woch.,  1906. — Hoff- 
meister's  Beitr.,  11  (1909). 

2  B.  T.  Barker:    Rockefeller  Institute,  8  (1908). 

3  S.  G.  Hedin:  Zeitschr.  f.  physiolog.  Chem.,  32  (1901). — C.  M. 
Takamura:  "bid.  63  (1909). — T.  B.  Leathes:  Journ  of  Physiol.,  28 
(1902). 

8 


114  ENZYMES 

gration  of  protein  has  been  observed  in  the  thymus,1 
which  is  rich  in  leucocytes.  In  leukemia,  the  spleen  and 
lymphatic  glands  may  be  enormously  enlarged,  and  in 
such  cases  it  has  been  observed  that  strong  proteolytic 
enzymes  split  the  proteins  of  the  spleen  or  the  glands. 
The  increased  quantity  of  the  enzymes  permits  a  study  of 
their  action,  which  is  identical  with  that  of  erepsin  or 
trypsin.  Proteins  are  converted  first  into  peptones,  and 
then  into  amino-acids.2 

Blood  yields  perhaps  a  small  quantity  of  arginase.3 

1  Fr.  Kutscher:    Zeitschr.  f.  physiolog.  Chem.,  34  (1901). 

2  O.  Schumm:    Hofmeister's  Beitrage,  7  (1906). 

3  A.  Kossel  and  H.  D.  Dakin:    ibid.,  42  (1904). 


CHAPTER   XVII 

Proteolytic  Enzymes  of  Tissues 

Salkowski 1  and  Jacoby 2  have  described  an  auto- 
digestion  or  autolysis  of  liver  and  other  organs.  If  we 
allow  liver  extracts  to  stand  for  several  days  or  weeks  at 
the  temperature  of  the  body,  a  part  of  the  proteins  is 
split  and  converted  first  into  peptones,  and  then  into 
amino-acids.  Since  the  first  publication  of  Jacoby,  much 
work  has  been  devoted  to  the  study  of  autolysis.  It  was 
thought  these  observations  threw  light  on  the  processes  of 
the  intermediate  metabolism  of  proteins,  because  earlier 
observations  had  shown  that  the  body  and  all  organs  lose 
protein  in  starvation  and  during  fever,  and  that  the  tissue- 
protein  can  be  drawn  upon  in  metabolism.  But  Vernon  3 
demonstrated  that  the  proteolytic  enzymes  of  the  kidney, 
liver,  and  other  organs  resemble  erepsin.  They  rapidly 
convert  peptones  into  products  not  giving  the  biuret  re- 
action, but  they  attack  tissue-proteins  only  very  slowly 
and  incompletely.      Later,  Abderhalden,4  using  artificial 

1  E.  Salkowski:    Zeitschr.  f.  klin.  Med.,  17  Suppl.  (1891). 

2  M.  Jacoby:  Zeitschr.  f.  physiolog.  Chem.,  30  (1900);  33  (1901). — 
Hofmeister's  Beitr.,  3  (1903). — S.  G.  Hedin  and  S.  Rowland:  Zeitschr. 
f.  physiolog.  Chem.,  32  (1901). 

3H.  M.  Vernon:    Journ.  of  Physiol.,  32  (1904);  33  (1905). 
4  E.  Abderhalden  (and  collaborators) :    Zeitschr.  f.  physiolog.  Chem., 
49,  5i>  53.  55  (1906-1908). 

115 


116  ENZYMES 

peptids,  confirmed  the  erepsin-like  character  of  the  tissue- 
enzymes.  The  ereptic  tissue-enzymes  are  not  identical 
with  the  blood-enzymes,  because  they  are  found  in  organs 
completely  freed  from  blood.1  This  erepsin  is  most  abun- 
dant in  the  kidney  (sometimes  present  in  larger  quantity 
than  in  the  intestinal  mucosa),  and  less  abundant  in  the 
liver  and  the  spleen;  still  less  in  the  lungs  and  brain,  and 
skeletal  muscles  yield  only  traces. 

A  second  tissue-enzyme  is  arginase,2  already  cited  as 
a  digestive  enzyme  of  the  small  intestine.  According  to 
Kossel  and  Dakin,  most  arginase  is  found  in  the  liver,  less 
in  the  kidney,  small  intestine,  and  thymus  and  lymphatic 
glands.     Spleen  and  muscles  do  not  seem  to  yield  arginase. 

The  function  of  tissue-erepsin  and  tissue-arginase  is 
not  certain.  They  either  have  a  function  in  the  metabo- 
lism of  the  liver  and  kidney;  a  view  which  is  supported  by 
Jacoby,  and  especially  by  Vernon,  who  designates  the 
ereptic  power  .of  tissues  as  a  measure  of  functional  capac- 
ity (and  the  failure  of  erepsin  in  muscle  recalls  the  fact 
that  the  working  muscle  does  not  burn  protein,  but  burns 
carbohydrates  and  fats) — or,  erepsin  and  arginase  attack 
those  peptones  and  arginine  in  the  liver  which  have  es- 
caped the  enzymes  of  the  intestine  in  the  course  of 
absorption.  There  are  some  peptones,  if  injected  subcu- 
taneously,  without  the  intervention  of  the  alimentary  canal, 
which  are  retained  in  the  organism  and  converted  into 

1  E.  Bloch:  Biochem.  Zeitschr.,  21  (1909). — O.  Cohnheim  and  D. 
Pletnew:    Zeitschr.  f.  physiolog.  Chem.,  68  (1910). 

2  A.  Kossel  and  H.  D.  Dakin:    ibid.,  41,  42  (1904). 


PROTEOLYTIC   ENZYMES    OF   TISSUES  117 

urea  and  carbonic  acid  in  the  ordinary  way.1  The  erepsin 
of  the  kidney  might  be  high  because  similarly  retained  by 
that  organ  though  destined  for  elimination. 

Our  knowledge  of  the  proteolytic  enzymes  of  the  tissues 
and  their  function  in  metabolism  is  unsatisfactory.  Some 
investigators  have  studied  the  enzymes  of  cancers  2;  they 
believe  that  malignant  tumors,  while  rapidly  increas- 
ing, push  away  the  tissues  as  an  abscess  does,  and 
they  have  tried  to  detect  enzymes  in  such  cancers.  But 
the  cancer  grows  in  another  way  than  does  the  softening 
abscess,  and  the  differences  found  between  normal  tissue 
and  cancer  are  so  small  that  they  can  be  easily  explained 
by  the  amount  of  blood  and  the  well-known  inflammations 
in  the  neighborhood  of  tumors.  It  would  be  interesting 
to  look  for  enzymes  in  connective  tissue.  When  we  see 
that  in  pregnancy  the  tissues  are  loosened  in  a  remarkable 
way,  we  might  suppose  that  proteolytic  enzymes  are 
fundamentally  responsible  for  the  process. 

There  are  examples  in  the  lower  animals  of  a  rapid 
solution  of  tissue  under  special  conditions.  The  dis- 
appearance of  the  tail  of  the  gnat  or  the  chewing  apparatus 
of  the  caterpillar  are  such  examples.  In  some  of  these 
cases,  Metchnikoff  and  his  pupils  have  observed  an  ener- 
getic phagocytosis;  the  enzymes  of  leucocyte  dissolves  the 

1  R.  Neumeister:  Zeitschr.  f.  Biol.,  24  (1888). — H.  Friedenthal  and 
M.  Lewandowski:  Arch.  f.  (Anat.  u.)  Physiolog.,  1899.  Suppl. — L.  B. 
Mendel  and  E.  W.  Rockwood:  Amer.  Journ.  of  Physiology,  12  (1904). — 
F.  P.  Underhill:    ibid.,  9  (1903). 

2  E.  Abderhalden,  Medigreceanu,  and  Pinkussohn:  Zeitschr.  f.  phy- 
siolog. Chem.,  66  (1910). 


118  ENZYMES 

organs  which  have  become  needless.  But  in  other  cases 
no  phagocytosis  is  seen,  and  the  solution  of  the  solid 
tissues  must  be  effected  by  proteolytic  ferments  which  are 
produced  or  activated  in  the  tissues  as  they  are  needed. 
The  testicles  of  the  salmon  grow  in  starving  animals,  and 
the  protein  is  derived  from  the  muscles.  Protein  of 
muscles  and  testicles  are  widely  different,  but  according 
to  Kossel  and  Weiss,1  proteins  of  muscles  yield  enough 
arginine  and  lysine  to  be  converted  into  testicle-protamine, 
and  a  proteolytic  enzyme  in  the  muscles  would  explain 
the  process.  Also  in  pregnancy,  it  seems  that  the  proteins 
of  the  mother  constitute  the  material  which  is  used  by  the 
growing  embryo,  and  therefore  the  tissues  of  the  mother 
must  be  dissolved.  But  the  proteolytic  ferments  which 
provoke  this  autolysis  are  not  known. 

1  A.   Kossel:     Zeitschr.   f.   physiolog.   Chem.,  44  (1905). — F.  Weiss: 

ibid.,  52  (1907). 


CHAPTER  XVIII 

Other  Hydrolytic   Enzymes   of   the   Blood 
and  Tissues 

Leucocytes  probably  contain  diastase,  because  we  can 
see  within  them  microscopical  granules  giving  the  color  re- 
actions of  glycogen.  After  clotting,  blood  serum  yields  dia- 
stase *  in  small  amount,  probably  derived  from  leucocytes. 
The  tissues,  and  especially  the  liver,  are  much  richer 
in  diastase.  In  the  liver-cells  it  lies  inside  of  the  proto- 
plasm, and  here  the  diastase  must  act.  This  is  the  reason 
that  the  extraction  of  diastase  from  the  liver  is  difficult, 
and  the  amount  of  enzyme  seems  to  be  small.  A  real 
difference  between  the  carbohydrate-splitting  enzymes  of 
the  alimentary  canal  and  those  of  the  tissues  is,  that  in  the 
tissues,  diastase  and  maltase  always  occur  in  the  same 
place,  and  cannot  be  separated.  Glycogen  is  thus  con- 
verted into  glucose.  Diastase  and  maltase  are  found  in 
many  or  all  organs,  which  is  in  harmony  with  the  general 
occurrence  of  glycogen.  They  are  found  in  great  amount 
in  the  liver,  in  muscles,  and  in  the  placenta.  As  to  the 
iiastase  of  the  liver,  Bang  2  has  reported  that  the  quantity 
found  in  pieces  of  the  organ  depends  upon  the  behavior 

1  C.  Hamburger:  Pfliiger's  Arch.,  60  (1895). — E.  Fischer  and  Nie- 
bel:  Berliner  Akad.  d.  Wissenschaften,  1896. — W.  Kiihne:  Heidel- 
berger  Naturhist.-med.  Verein,  N.  F.  (1877). 

2 1.  Bang:    Hofmeister's  Beitr.,  10  (1908). 

119 


120  ENZYMES 

of  the  animal  immediately  before  death,  and  that  it  can 
be  changed  by  nervous  stimulation,  by  the  activity  of  the 
liver,  etc.  Other  carbohydrate-splitting  enzymes  in  the 
body  are  not  known. 

Whether  blood  and  blood  corpuscles  contain  lipase  in 
traces  or  in  great  amount,  is  questioned.1  In  the  tissues, 
lipase  is  found  in  large  quantity  in  the  liver.  It  is  identical 
with  the  pancreatic  lipase,  or  resembles  it  closely,  because 
it  is  activated  by  the  bile,  and  acts  best  in  a  neutral  medium 
(vide  supra).  According  to  Nencki,2  muscle  and  kidney 
contain  weak  lipases;  the  other  tissues,  especially  the 
adipose  tissue,  have  not  been  investigated. 

Some  tissues  contain  nuclease,  the  same  enzyme  which 

occurs  in  the  pancreas  and  the  small  intestine,  but  it  is 

not  determined  whether  the  enzyme  originates  in  the  cells 

of  the  tissues,  or  comes  from  the  leucocytes.     It  is  found 

in  greatest  amount  in  the  spleen  3  and  the  thymus,4  organs 

rich  in  leucocytes. 

That  tissues  contain  lecithases  is  not  astonishing,  be- 

f 
cause   the   steapsins   dissociate  lecithin;   thus   choline  is 

found  among  the  products  of  autolysis  of  the  liver  and 
other  organs.  According  to  Sieber,  lipase  of  the  blood- 
serum  does  not  attack  lecithin. 

1 C.  Schumow-Simanowski  and  N.  Sieber:  Zeitschr.  f.  physiolog. 
Chem.,  49  (1906). — W.  Connstein:  Ergebnisse  d.  Physiologie,  337,, 
Biochemie  (1904). 

2  (M.  Nencki  and)  E.  Liidy:  Arch.  f.  exper.  Path.  u.  Pharm.,  25 
(1889). 

3  O.  Schumm:    Hofmeister's  Beitr.,  7  (1906). 

4  Fr.  Kutscher:    Zeitschr.  f.  physiolog.  Chem.,  34  (1901). 


CHAPTER  XIX 

Urease  and  Nucleases 

These  enzymes  break  the  connection  between  nitrogen 
and  carbon  with  the  entrance  of  water,  as  do  the  hydro- 
lytic  enzymes,  but  differ  greatly  from  the  proper  hydro- 
lytic  enzymes.     They  bring  about  the  following  reactions: 

H2NCNH2  +  2H20  =  C03H2  +  2NH3 

Carbonic  (Urease) 

O  Acid     Ammonia 

Urea 

N=C  NH2  HN— CO 

I    !  !     I 

HC    C— NH  +  H20  =  HC     C— NH  +  NH3    (Adenase) 

II      II        >CH  II      II        >CH 

N— C— INT  N— c— w 

Adenine  Hypoxanthine 

HN— CO  HN— CO 

H2NC     C— NH  OC     C-NH     (Guanase) 

||      ||        \CH  +  HoO  =     |       ||        \CH 

N— C— N^  H— C— N^ 

Guanine  Xanthine 

Urease  is  found  in  many  bacteria  which  provoke  fer- 
mentation of  urine  eliminated  from  the  body,  or,  in 
pathological  cases,  within  the  bladder.  It  has  not  been 
observed  in  the  organs  of  the  animal  body.     According  to 

Miquel,  we  can    extract  and  separate  urease  from  the 

121 


122  ENZYMES 

bodies  of  the  bacteria.1  Adenase  and  guanase  were  first 
found  in  yeast  by  Lehmann  2  in  Kossel's  laboratory.  In 
recent  years  they  have  been  found  in  the  liver,  kidney,  and 
other  organs  and  investigated  by  Schittenhelm  3  and  Jones  4 
and  their  collaborators.  They  can  be  easily  dissolved. 
According  to  Schittenhelm,  xanthine  is  converted  by  this 
enzyme  in  the  presence  of  abundant  oxygen  into  uric  acid, 
showing  that  under  these  conditions  the  enzyme  provoked 
an  oxidation  instead  of  an  hydrolysis.  Thus  the  purin- 
bases,  adenine  and  guanine,  cleavage-products  of  nucleic 
acid  split  by  nucleases,  are  converted  into  the  oxypurins, 
and  possibly  they  may  be  oxidized  further  into  uric  acid, 
one  of  the  regular  waste  products  of  the  mammalians. 
In  other  mammals,  for  instance  the  dog,  uric  acid  formed 
in  this  or  some  other  manner,  is  converted  by  a  further 
"uricolytic"  enzyme  into  allantoin.5 

Uric  acid  undergoes  the  same  oxidation  when  acted  on 
by  a  mild  oxidizing  agent,  such  as  potassium  permangan- 

HN  -  CO 

|  HN  -  CO 

OC        C  -  NH  | 

|         ||        >  CO  +  02  =  OC  NH2 

HN-C-NH  >CO  +  C02 

HN  -  CH  -  NH 

Uric  acid  Allantoin 

1  Miquel:     Compt.  rend.,  in  (1890). 

2  V.  Lehmann:    Zeitschr.  f.   physiolog.  Chem.,  9  (1885);    cf.  K.  Shiga, 
ibid.,  42  (1904). 

3  A.  Schittenhelm:    ^^.,42,43(1904);    45,46(1905);    48,50(1906). 
*  W.  Jones:    ibid.,  41,  42,  44,  48. 

5  W.  Wiechowski:    Hofmeister's  Beitr.,  9  (1906). 


UREASE   AND   NUCLEASES  123 

ate.  In  other  animals,  like  man,  allantoin  is  formed  and 
eliminated,  but  nevertheless  a  uricolytic  enzyme  capable 
of  destroying  uric  acid  has  been  extracted  from  the  organs 
of  man,  the  ox,  and  pigs,  by  Wiechowski,  Jones,  and 
Schittenhelm.  It  is  not  known  how  this  enzyme  acts; 
whether  there  is  a  stronger  oxidation  or  no  oxidation. 

It  seems  that  similar  aminolytic,  or  desaminating, 
enzymes,  which  replace  the  NH2  group  by  the  OH  group, 
are  widely  distributed  in  organs.  The  specificity  of  these 
enzymes  is  not  very  evident.  Schittenhelm  supposes  that 
only  one  enzyme  attacks  adenine  and  guanine;  Jones  sug- 
gests the  existence  of  two  or  three  enzymes,  and  it  is  not 
impossible  that  the  same  enzyme  which  attacks,  for  in- 
stance, alanine,  and  converts  it  into  lactic  acid,  attacks 
likewise  adenine  and  guanine.  If  this  is  so,  we  must  be 
very  slow  to  draw  conclusions  regarding  specificity,  because 
we  cannot  know  that  an  enzyme  studied  is  destined  for 
the  special  reaction  which  we  employ  it  to  provoke. 


CHAPTER   XX 


The  Oxidases 


Food  is  burned  in  and  by  the  living  protoplasm,  and 
thus  supplies  the  energy  needed  for  life.  This  combustion 
could  be  an  anoxybiotic  process,  but,  in  the  higher  animals, 
combustion,  which  takes  place  continuously  in  the  tissues, 
is  an  oxidation,  and  attempts  have  for  a  long  time  been 
made  to  detect  and  extract  enzymes  which  oxidize  sub- 
stances, especially  sugar  or  fat.  Some  enzymes  have  been 
found  which  convert  aldehydes  into  acids,  or  produce 
similar  reactions,  and  act  only  in  the  presence  of  oxygen. 
Other  substances  have  been  discovered,  which  liberate 
oxygen  from  hydrogen  peroxide,  and  the  oxygen  thus  set 
free  can  produce  oxidative  reactions.  Whether  in  these 
last  substances  we  are  dealing  with  enzymes  or  with 
agencies  of  a  wholly  different  type,  is  not  certain.  The 
facts  and  observations  described  by  the  different  authors 
have  not  been  generally  accepted.  Though  the  study  of 
such  enzymes  is  of  great  interest,  a  good  deal  of  the  ex- 
perimental work  has  been  very  inexact.  It  is  important 
that  we  separate  the  main  metabolism-enzyme  of  the  type 
of  zymase  from  the  so-called  oxidases.  The  latter  group 
of  substances  can  now  be  briefly  reviewed. 

i.  Catalase.     Schonbein  found  in  1863,  that  hydrogen 

peroxide  is  decomposed  into  oxygen  and  water  by  platinum 

black  and  other  inorganic  substances,  and  likewise  by 

124 


THE    OXIDASES  125 

blood,  milk,  tissues,  etc.  In  the  last  few  years,  Bredig 
and  his  pupils  have  studied  the  decomposition  of  hydrogen 
peroxide  by  organic  and  inorganic  catalysts,  and  have 
tried  to  support  Schonbein's  view,  that  this  decomposition 
by  colloidal  platinum,  gold,  or  mercury,  resembles  enzyme 
action.  Many  finely  divided  substances,  especially  the 
colloidal  solutions  of  the  metals  platinum,  gold,  and 
mercury,  produce  this  decomposition.  Tissues  and  animal 
fluids  are  also  colloidal  solutions,  and  provoke  the  same 
reaction.  If  the  colloidal  properties  of  the  tissues  or  the 
blood  are  destroyed  by  heating,  they  lose  the  capacity  of 
decomposing  hydrogen  peroxide.  Because  enzymes  are 
destroyed  by  heating,  there  seems  to  be  a  resemblance  be- 
tween inorganic  catalysts  and  enzymes.  But  no  further 
proof  has  been  brought  forward  that  the  two  processes 
are  of  a  similar  character.  Hydrogen  peroxide  never 
occurs  in  our  bodies,  and  I  do  not  believe  that  the  "cata- 
lase"  is  an  enzyme.1 

2.  The  following  reactions  are  more  or  less  well  defined 
and  cleared  up,  and  there  can  be  no  doubt  that  they  are 
of  enzymotic  nature.  The  oxidases  oxidize  substances 
readily  oxidizable  in  the  presence  of  atmospheric  oxygen. 

a.  Aldehydase.2  This  converts  salicylaldehyde  into 
salicylic  acid: 


1  G.  Bredig:  Ergebnisse  d.  Physiologie,  I.  Biochemie  (1902);  M. 
Jacoby:  ibid.,  I.  Biochemie  (1902). — A.  Bach  and  R.  Chodat:  Biochem- 
isches  Zentralblatt,  i.  (1903);  viii.  (1909). — Raudnitz:  Zentralbl.  f. 
Physiologie,  12  (1899). — Spitzer:    Pfltiger's  Arch.,  67  (1897). 

2  M.  Jacoby:    Ergebnisse  d.  Physiologie,  I.  Biochemie  (1902). 


126  ENZYMES 

/OH  (ortho)  yOH  (ortho) 

xCHO  xCOOH 

or  benzylaldehyde  into  benzoic  acid :  C6H5CHO  +  O  = 
C6H5COOH:  or,  formaldehyde  into  formic  acid; 
HCHO  +  O  =  HCOOH,  and  other  similar  reactions. 
Salicylic  acid  can  be  detected  and  estimated  easily 
by  the  red  color  reaction  with  ferric  chloride,  and 
after  adding  salicylaldehyde,  the  red  color  occurs 
in  many  tissues,  especially  in  the  liver,  spleen,  kidney, 
and  suprarenal  glands.  Blood  gives  the  reaction 
to  a  slight  extent.  Jaquet,1  Abelous  and  Biarnes 2 
and  Salkowski,3  have  demonstrated  that  the  oxidation 
of  salicylaldehyde  is  effected  by  an  enzyme,  which  can 
be  extracted  from  the  tissues,  and  Jacoby 4  has  freed 
the  oxidizing  enzyme  from  proteins  and  most  other  im- 
purities. The  aldehydase  of  liver  studied  by  him,  is 
an  enzyme,  the  solubility  and  precipitability  of  which 
are  best  known,  and  my  description  of  the  chemical 
properties  of  enzymes  is  based  chiefly  on  Jacoby' s 
work  on  aldehydase.  He  obtained  a  clear,  aqueous 
solution,  which  could  convert  salicylaldehyde  into  salicylic 
acid  in  a  short  time  and  in  great  amounts.  The  alde- 
hydase is   very  resistant    to  heat   and    other    influences, 

1  Jacquet:    Arch.  f.  exper.  Path.  u.  Pharmakol.,  29  (1892). 

2  Abelous  and  Biarnes:    Arch,  de  Physiol,  et  de  Path,  gen.,  1894,  1898. 

3  E.  Salkowski:     Virchow's  Arch.,  147  (1898);'  Zeitschr.    f.     physiol. 
Chem.,  13  (1889). 

4  M.   Jacoby:     Zeitschr.   f.   physiolog.   Chem.,  30  (1900). — A.   Med- 
wedew:    Pniiger's  Arch.,  81  (1900). 


THE    OXIDASES  127 

and  is  destroyed  only  at  8o°  C.  It  is  resistant  to  alka- 
lies, but  acids  destroy  it.  It  is  possible,  but  not  quite 
certain,  that  formaldehyde  and  salicylaldehyde  are  oxi- 
dized by  the  same  enzyme.  As  Jacoby 1  has  pointed 
out,  the  embryos  of  young  pigs  contain  no  aldehydase, 
while  the  adults  do. 

b.  Laccase.  This  has  been  detected  and  especially 
studied  by  the  French  physiologist,  Bertrand.2  It  con- 
verts hydroquinone  into  quinone: 

.OH  JO 

2C6H4<f        +  02  =  2C6H4<f  I   +  2H20. 
XOH  X0 

It  converts  pyrogallic  acid,  with  a  simultaneous  combina- 
tion of  three  molecules  into  one,  forming  f urf urogallin : 

,       x  /O.OC6H3(OH)2 

3C6H3  (OH),  +  02=  HOC6H3<  '2+  2H20 

\).OC6H3(OH)2 

It  likewise  oxidizes  and  condenses  gallic  acid  and  other 
phenols  into  the  corresponding  quinones. 

The  compounds  formed  by  laccase  are  brown  or  black, 
and  the  action  of  the  laccase  can  be  recognized  by  the 
rapid  change  of  color  into  brown  or  black  in  the  presence 
of  air.  Among  these  reactions  is  the  oxidation  of  urushic 
acid  or  laccol  into  oxyurushic  acid : 

1  M.  Jacoby:    Zeitschr.  f.  physiolog.  Chem.,  33  (1901). 

2  G.  Bertrand:  Compt.  rend.,  118,  120,  123,  124;  Compt.  rend,  de  la 
Soc.  de  Biol.,  121  (1897-1898). — B.  Slowtzoff:  Zeitschr.  f.  physiolog. 
Chem.,  31  (1900). 


128  ENZYMES 

This  reaction  was  the  first  fermentative  oxidation  to  be 
investigated,  and  led  to  the  discovery  of  laccase.  The 
sap  of  the  East  Asian  lac  tree  contains  laccase  and  urushic 
acid,  or  laccol,  and  the  laccase  converts  urushic  acid  into 
oxyurushic  acid,  as  was  observed  by  Yoshida  in  1893  and 
explained  by  Bertrand  in  1894.  The  product  of  the  con- 
version is  the  insoluble  black  oxyurushic  acid,  which  thus 
gives  rise  to  the  brilliant  black  lustre  of  the  lacquer  manu- 
factured in  Japan  and  China.  But  the  laccase  has  been 
found  also  in  many  plants,  such  as  the  roots  of  the  beet, 
carrot,  and  turnip;  in  the  potato,  apple,  and  pear,  in 
clover  and  asparagus,  in  germinating  seeds,  in  some 
flowers,  and  in  many  fungi.  Laccase  is  associated  with 
manganese,  which  is  its  activator  or  co-ferment  (vide 
supra) . 

c.  Tyrosinase.1  This  enzyme  resembles  laccase  in 
action  and  occurrence,  but  is  not  associated  with  man- 
ganese. It  converts  phenols,  like  xylenol,  amido-phenols, 
indophenol,  phenolphtalein,  naphthol,  and  similar  com- 
pounds, into  substances  richer  in  oxygen.  In  most  cases, 
these  substances  are  at  the  same  time  combined  to  form 
larger  molecules.  For  instance,  amidophenol  and  phenol 
are  oxidized  and  combined  to  form  indophenol. 

X6H4-0 
C6H5OH  +  CeH4(NH2)(OH)+0  =  HN<(  I   +  H20. 

Indophenol  has  a  blue  color;  the  other  products  formed 

1 H.  Steudel:  Deutsche  med.  Woch.,  1900. — M.  Gonnermann: 
Pfluger's  Arch.,  89  (1902). 


THE    OXIDASES  129 

by  the  tyrosinase  are  brown  or  black.  The  enzyme  has 
received  its  name  because  it  seems  to  act  upon  the  cleavage 
product  of  proteins,  tyrosine,  which  is  a  phenol.  If  we 
add  a  tyrosinase  solution  to  tyrosine,  it  becomes  first  red, 
later  black,  and  then  forms  a  black  precipitate.  But 
according  to  E.  Schulze,1  extracts  of  plants,  which  are 
acted  upon  by  tyrosinase,  do  not  always  contain  tyrosine. 
It  is  possible,  therefore,  that  it  is  not  tyrosine  that  is  oxi- 
dized, but  an  impurity  which  often  accompanies  the 
tyrosine  obtained  from  proteins.  Until  the  relations  are 
completely  cleared  up,  however,  it  will  be  better  not  to 
change  the  name  of  the  enzyme. 

Tyrosinase  occurs,  like  laccase,  in  many  plants,  especial- 
ly in  fungi,  for  instance,  in  Russula  and  Argaricus,  which 
are  the  best  objects  for  demonstrating  oxidases.  In 
animals  it  occurs  in  the  blood  or  in  the  hemolymph  of 
certain  butterflies  and  other  insects,  in  the  intestinal  juice 
of  the  meal  worm,  in  the  larva  of  Tenebrio  molites,  in  the 
crayfish,  and  other  higher  and  lower  animals,  v.  Furth 
and  Schneider  2  have  observed  that  the  black  precipitate 
formed  by  tyrosinase  resembles  in  properties,  cleavage- 
products,  and  in  composition,  the  melanins  naturally  oc- 
curring in  the  skin,  hair,  and  other  organs,  and  in  the  ink- 
bag  of  the  Sepia;  and  v.  Furth  has  also  shown  that  the 
ink-gland  and  the  ink-bag  of  the  Sepia  contain  much 
tyrosinase,  which  might  perhaps  explain  the  formation  of 
the  ink-melanin  in  the  Sepia.     The  three  oxidases  also 

1  E.  Schulze:    Zeitschr.  f.  physiolog.  Chem.,  50  (1906). 

2  O.  v.  Furth  and  H.  Schneider:    Hofmeister's  Beitr.,  1  (1901). 

9 


130  ENZYMES 

convert  guaiaconic  acid,  C20H24O5,  into  guaiacum  blue, 
C20H22O6.  The  guaiac  test,  the  blue  color  with  guaiac 
tincture  or  the  pure  acid,  is  a  very  easy  and  convenient 
test  for  oxidases,  and  is  often  used  by  investigators. 

The  differences  between  the  individual  oxidases  are  not 
well  understood.  Enzymes  associated  with  manganese 
must  be  of  an  individual  type.  And  I  think  we  are  right 
in  separating  the  aldehydases  converting  aldehydes  into 
acids,  from  the  tyrosinase,  which  on  the  contrary  forms 
aldehyde-like  compounds.  It  must  be  mentioned  that 
salicylaldehyde,  pyrogallol,  etc.,  do  not  occur  in  the 
organisms;  in  using  them  we  are  not  working  with  the 
natural  substrate  for  the  oxidases,  and  the  evidence  that 
an  individual  extract  attacks  only  aldehydes,  or  only 
hydroquinone,  or  only  gives  the  guaiacum-blue  test,  is 
not  very  conclusive.  The  question  as  to  the  number  of 
oxidases  remains  open. 

The  compounds  which  are  acted  upon  by  the  three 
oxidases  are  readily  oxidizable  in  an  alkaline  medium, 
and  absorb  oxygen  even  without  oxidases.  Hydroquinone, 
pyrogallol,  and  amidophenol  are  used  in  photography 
as  reducing  substances;  and  pyrogallol  is  also  employed  in 
gas  analysis  because  of  its  oxygen-absorbing  properties. 
The  oxidases  seem  only  to  accelerate  the  oxidation,  and 
enable  it  to  take  place  in  a  weakly  alkaline  medium;  while 
without  enzymes,  oxidation  requires  more  alkali  and  more 
time.  The  oxidases  are  oxygen  bearers  or  carriers,  and 
the  view  regarding  enzymes  as  mere  catalysts  is  based 
upon  the  properties  of  these  oxidases. 


THE    OXIDASES  131 

The  presence  of  oxygen  is  necessary  for  the  action  of 
oxidases,  and  reactions  are  best  observed  with  a  current 
of  air  passing  through  the  reaction-flasks.  For  the  action 
of  laccase  associated  with  manganese,  Bertrand  suggests 
a  scheme  to  represent  the  carrying  of  oxygen  in  a  water- 
solution  from  the  air  to  the  substrate  of  the  enzyme. 

R"Mn  +  H20    =  R"H2  +  MnO. 

MnO  +  02        =  MnOs  +  O. 

R"H2  +  Mn02  =  R"Mn  +  H,0  +  O. 

Thus  after  the  reaction,  the  solution  contains  the  enzyme 
R"  in  an  unchanged  state,  and  O  not  as  an  inactive  mole- 
cule, but  as  free  uncombined  oxygen  in  a  nascent  state. 
The  action  of  the  laccase,  according  to  this  theory,  would 
be  to  convert  the  inactive  oxygen  of  the  air  into  the  more 
energetic  form.  Aldehydase  and  tyrosinase  do  not  con- 
tain manganese,  which  is  never  found  in  the  body  of  higher 
animals.  Spitzer  has  observed  that,  in  solubility,  the 
oxidases  agree  with  nucleoproteins.  In  some  nucleo- 
proteins,  iron  was  observed,  and  Spitzer  has  supposed  that 
in  these  oxidases  manganese  is  replaced  by  iron.  The 
supposition  is  rejected  by  Sauerland  x  and  Masing,2  who 
failed  to  find  any  iron  in  nucleic  acid  and  in  the  eggs  of 
the  sea-urchin,  which  undergo  a  strong  oxidation  during 
development.  Jacoby 3  separated  aldehydase  from  nucleo- 
proteids  without  loss  of  oxidizing  power.     Nevertheless, 

1  F.  Sauerland:    Zeitschr.  f.  physiolog.  Chem.,  64  (1909). 

2  E.  Masing:  ibid.,  66  (19 10). 

3  M.  Jacoby:    ibid.,  30  (1900);  33  (1901). 


132  ENZYMES 

it  seems  that  there  is  some  association  between  oxidases  and 
nucleoproteins,  for  we  have  seen  that  the  hydrolytic 
enzymes,  like  pepsin,  are  not  nucleoproteins,  but  in  or- 
ganisms are  always  associated  with  them.  Warburg  and 
Morawski *  have  observed  that  the  absorption  of  oxygen 
by  red  corpuscles  is  closely  connected  with  the  occurrence 
of  nucleic  acid  in  the  corpuscles,  though  the  nucleus  as  a 
visible  structure  is  not  present. 

Another  scheme  of  reaction  for  oxidases  and  for  oxida- 
tions in  protoplasm,  was  given  years  ago  by  Hoppe-Seyler  2 
and  Bunge,3  and  in  recent  years  by  Bach  and  Chodat,4 
Kastle  and  Loevenhart,5  and  Dakin.6  These  investigators 
have  supposed  that,  in  tissues,  readily  oxidizable  sub- 
stances are  formed,  which  have  a  special  capacity  for 
absorbing  oxygen.  By  these  substances  the  oxygen- 
molecule,  02,  is  dissociated  into  two  atoms;  one  atom  is 
absorbed,  and  the  other,  in  the  nascent  state,  can  attack 
other  less  oxidizable  compounds.  Hoppe-Seyler  and 
Bunge  did  not  discuss  the  chemical  nature  of  the  readily 
oxidizable  substances;  Bunge  mentioned  only  the  fact 
that  cuprous  and  ferrous  salts  have  this  property,  which 
he  assumed  for  the  tissues. 


1  O.  Warburg:    Zeitschr.  f.  physiolog.  Chem.,  59  (1909). — P.  Moraw- 
ski:   Arch.  f.  exper.  Path.  u.  Pharmak.,  60  (1909). 

2  F.  Hoppe-Seyler:    Zeitschr.  f.  physiolog.  Chem.,  2  (1878);  10  (1886). 

3  G.  Bunge:    " Physiologische  Chemie."     Leipzig,   1901. 

4  A.  Bach  and  R.  Chodat:    Biochem.  Zentralbl.,  1  (1903);   8  (1909). 

6  A.  S.  Loevenhart  and  J.  H.  Kastle:  Amer.  Chem.  Journal,  29  (1903). 

A.  S.  Loevenhart:    Amer.  Journ.  of  Physiology,  13  (1905). 
6  H.  D.  Dakin:    Journ.  of  Biolog.  Chemistry,  4  (1908) 


THE    OXIDASES  133 

Bach  and  Chodat,  Kastle  and  Loevenhart,  and  Dakin, 
suppose  the  intermediate  formation  of  peroxides  not  known 
in  the  time  of  Hoppe-Seyler.  Bach  and  Chodat  have 
described,  besides  the  oxidases  mentioned,  another  type, 
the  peroxidases,  which  give  the  guaiac-blue  test  only  in 
the  presence  of  hydrogen  peroxide  or  sodium  peroxide. 
Experimental  proofs  for  these  latter  compounds  are  not 
very  conclusive,  and  I  think  that  the  whole  theory  of 
oxidation  by  reduction  and  reoxidation  of  substances 
which,  in  this  way,  act  as  oxygen  carriers  is  not  a  satis- 
factory explanation  of  the  process  as  it  takes  place  in  the 
body. 

It  is  true  that  in  addition  to  the  oxidation  of  foods,  there 
are  reducing  processes  in  the  organism  such,  e.g.,  as  the 
conversion  of  atoxyl  into  arsenophenylglycine,  recently 
demonstrated  by  Ehrlich  *;  or  the  reduction  of  methylene 
blue  in  the  tissues  immediately  after  death,  which  was 
used  by  Ehrlich  2  as  a  measure  of  the  avidity  of  tissues  for 
oxygen;  or  the  conversion  of  acetoacetic  acid  into  oxy- 
butyric  acid, 

CH3.CO.CH2COOH  &  CH3CHOH.  CH2 .  COOH, 

found  by  Blum,3  Friedmann  and  Maase,4  Dakin,5  and 

1  P.  Ehrlich:  Deutsche  dermatolog.  Ges.,  1908. — W.  Roth:  Ber- 
liner klin.  Woch.,  1909,  No.  11. 

2  P.  Ehrlich:     " SauerstofTbediirfniss  des  Organismus,"  Berlin,  1890. 

3  L.  Blum:    Munch,  med.  Woch.,  1910. 

4  E.  Friedmann  and  C.  Maase:    Biochem.  Zeitschr.,  26,  19 10. 

5  H.  D.  Dakin:  Journ.  Amer.  Med.  Assoc,  1910;  Munch,  med. 
Woch.,  1910. 


134  ENZYMES 

Neubauer.1     But    against    the    generalization    of    these 
processes  we  have  three  arguments. 

i.  Peroxides  are  never  found  in  the  organism,  and 
substances  which  could  serve  as  oxygen  carriers  occur  in 
too  small  amount  to  be  of  importance.  According  to  this 
theory,  we  are  not  dealing  with  enzymes,  which  can  convert 
an  enormous  quantity  of  matter;  but  one  equivalent 
weight  of  the  reducing  substance  can  liberate  oxygen  only 
for  one  equivalent  weight  of  the  less  oxidizable  substance, 
and  there  are  also  difficulties  from  the  point  of  view  of 
energetics. 

2.  Jacoby  has  purified  aldehydase  by  a  prolonged  treat- 
ment. If  aldehydase  were  a  compound  readily  oxidizable, 
it  must  have  undergone  oxidation  during  these  manipula- 
tions. He  observed,  also,  that  the  pure  solution  does  not 
convert  salicylaldehyde  according  to  equivalent  weights. 
He  allowed  the  process  to  go  to  completion  and  then 
removed  the  salicylic  acid  formed  by  dialysis,  and  added 
new  salicylaldehyde;  a  new  formation  of  salicylic  acid 
could  now  be  observed.     This  is  an  enzymotic  process. 

3.  All  the  theories  of  oxidation  by  intermediate  reduc- 
tion are  upset  by  the  discovery  of  zymase  and  the  enzymes 
resembling  it  which  form  lactic  or  acetic  acid.  These 
enzymes  are  found  in  expressed  juices,  which  do  not  con- 
tain reducing  substances,  and  which  do  not  oxidize  other 
compounds.  They  convert  in  a  specific  manner  only  those 
compounds  which  furnish  energy  for  the  vital  processes 

1  O.  Neubauer:    Zeitschr.  f.  physiolog.  Chem.,  70  (1910). 


THE    OXIDASES  135 

of  the  individual  micro-organism.  They  act  like  true 
enzymes,  that  is  to  say,  they  convert,  not  the  equivalent 
weight,  but  an  unlimited  quantity. 

Combustion  in  the  protoplasm  of  cells  or  tissues  is 
therefore  occasioned  by  enzymes,  and  not  by  another 
process,  such  as  the  formation  of  peroxides,  etc.  But  the 
combustion  of  sugar  by  zymase,  the  only  isolated  and  well- 
investigated  enzyme,  is  not  an  oxidation.  It  follows  the 
equation : 

C6H12Oe  =  2C03  +  2C2H5OH. 

Glucose  is  broken  down  into  alcohol  and  carbon  dioxide 
without  loss  or  addition  of  water,  or  addition  of  oxygen. 
Likewise,  the  formation  of  lactic  acid  is  not  an  oxidation, 
but  only  a  cleavage  of  the  molecule  of  sugar: 

C6H1206  =  2C3H603. 

Some  investigators,  for  instance  Bunge,  have  thought 
that  such  an  intramolecular  change  may  occur  generally 
in  the  tissues  and  give  rise  to  compounds  readily  oxidizable. 
Bunge  1  and  Weinland  2  have  observed  that  the  metabolism 
of  the  round  worm  is  a  mere  intramolecular  change  with- 
out oxidation;  glucose  is  converted  into  valeric  acid  and 
carbon  dioxide.  Also,  the  pupa  of  the  fly  has,  according 
to  Weinland,3  for  a  certain  period  of  metamorphosis,  an 
anoxybiotic  metabolism.  It  seems  possible  that  perhaps, 
in  higher  animals,  the  readily  oxidizable  substances  are 

1  G.  Bunge:    Zeitschr.  f.  physiolog.  Chem.,  14  (1890). 

2  E.  Weinland:    Zeitschr.  f.  Biologie,  42  (1901). 

3  Ibid.,  48  (1905). 


136  ENZYMES 

formed  in  the  tissues,  and  oxidized  later  in  the  blood  or 
lungs.  That  organs  and  the  expressed  juice  or  pulp  of 
organs  absorb  oxygen,  is  no  evidence  against  this  theory, 
because  we  cannot  always  separate  organs  and  blood,  and 
because  the  final  oxidation  could  occur  in  the  lymph,  and 
not  in  the  blood-vessels.  But  the  whole  theory  that  the 
food-stuffs  utilized  in  the  tissues  are  chiefly  split  and 
oxidized  only  in  a  secondary  process,  is  impossible  from 
the  energetical  point  of  view.  The  investigation  of  the 
so-called  respiratory  quotient,  the  relation  between  the 
absorbed  oxygen  and  the  eliminated  carbon  dioxide,  and 
the  calorimetric  investigations  of  Rubner  *  and  of  Atwater 
and  Benedict,2  have  shown  that  carbohydrates  and  fats 
are  as  completely  burned  in  the  organism  as  in  a  calori- 
meter bomb,  and  that  the  substances  deliver  the  same 
quantity  of  heat  as  in  artificial  combustion.  It  has  been 
shown  by  Barcroft 3  and  his  collaborators,  by  the  author,4 
and  by  Vernon,5  that  the  respiratory  quotient  of  the  in- 
dividual tissues  is  similar  to  or  the  same  as  the  respiratory 
quotient  of  the  whole  organism. 

It  was  shown  by  Frank,6  by  Zuntz,7  and  by  Atwater  and 

1  M.  Rubner:    Zeitschr.  f.  Biolog.,  21  (1885);   30(1892);   42(1901). 

2  W.  O.  Atwater  and  F.  G.  Benedict:     Carnegie  Institution,  1905. — 
F.  G.  Benedict:    Amer.  Journ.  of  Physiology,  24  (1909). 

3  J.  Barcroft:    Journ.  of  Physiology,  31,  32,  t,^  (1904-1907). 

4  O.  Cohnheim  and  D.  Pletnew:     Zeitschr.  f.  physiolog.  Chem.,  69 
(1910). 

5  H.  M.  Vernon:    Journ.  of  Physiology,  35,  36,  39  (1906-1909). 

6  O.  Frank:    Ergebnisse  d.  Physiologie,  3  (Biophysik),  1904. 

'  N.  Zuntz:    Pfliiger's     Arch.,     63  (1891);     68    (1897).— A.     Diirig: 
Pfliiger's  Arch.,  113  (1906). 


THE    OXIDASES  137 

Benedict,1  mat  a  third,  or  thirty  per  cent,  of  the  heat  pro- 
duced in  muscle,  is  converted  into  mechanical  energy  or 
work.  Frank  has  directly  measured  the  output  of  heat  in 
isolated  frog's  muscles,  using  a  delicate  thermo-electrical 
method.  The  weight  raised  by  the  muscles  gives  the  me- 
chanical energy.  In  the  best  experiments  this  was  one- 
half  of  the  heat  produced  at  the  same  time,  so  that  a  third 
of  the  energy  produced  became  work,  and  two-thirds 
became  heat. 

At  water  and  Benedict  have  determined  the  heat  given 
off  from  the  body  in  the  respiration  calorimeter.  The 
external  work  was  measured  by  a  specially  devised 
ergometer,  and  they  found  that  a  fifth  of  the  total  energy 
becomes  muscular  work. 

Zuntz  and  his  collaborators  have  measured  the  absorp- 
tion of  oxygen  during  a  climb,  and  with  the  heat  produced 
by  this  oxygen,  they  compared  the  weight  of  the  man  with 
the  height  reached  in  a  given  time.  They  found  that 
thirty  per  cent  of  the  total  energy  liberated  in  the  body 
during  muscular  work  becomes  mechanical  energy.  Since 
the  heart,  the  respiratory  muscles,  and  other  muscles  not 
directly  moving  the  body  absorb  an  increased  amount  of 
oxygen  in  climbing,  more  than  thirty  per  cent  of  the  whole 
energy  must  be  converted  into  movement  or  work.  In 
the  best  steam  engines  constructed  by  man,  a  much 
smaller  amount  of  the  total  potential  energy  of  the  fuel  is 
utilized  as  work,  not  more  than  ten  to  thirteen  per  cent, 
the  rest  being  lost  as  heat. 

1  hoc.  cit. 


138  ENZYMES 

If  the  quantitative  relation  of  work  to  heat  is  so  much 
better  in  the  muscle  engine  than  in  the  steam  engine,  we 
must  conclude  that  in  muscle  the  latent  potential  energy 
must  be  completely  or  to  a  very  great  extent  set  free  during 
combustion.  We  know  that  there  are  conversions  of 
sugar  without  oxidation  or  with  a  partial  oxidation,  as 
exemplified  in  the  conversion  into  ethyl  alcohol  and  carbon 
dioxide,  or  into  lactic  acid,  or  the  conversion  into  glycerin- 
aldehyde  or  methylglyoxal,  or  into  acetone  or  ethyl-  or 
formaldehyde.  Furthermore  we  observe  the  probable 
conversion  of  fatty  acids  into  oxybutyric  acid  and  acetone. 
But  all  these  conversions  liberate  only  a  small  amount  of 
the  energy  which  is  set  free  by  the  complete  combustion 
of  sugar  or  fat  into  water  and  carbon  dioxide.  The  follow- 
ing table  shows  in  large  calories  the  heat  produced  by  the 
complete  combustion  of  ioo  gm.  of  glucose  into  water 
and  carbon  dioxide,  and  by  the  partial  conversion  of  the 
sugar  into  ethyl  alcohol,  lactic  acid,  formic  acid,  glyoxylic 
acid,  glycolic  acid,  formaldehyde,  glycerinaldehyde,  glyoxal 
and  methylglyoxal.  The  figures  were  obtained  by  sub- 
tracting the  heat  of  combustion  of  these  compounds  from 
the  total  heat  of  combustion  of  the  sugar.  The  table  also 
gives  the  corresponding  figures  for  stearic  acid  and  three 
compounds  which  are  probably  derived  from  the  fatty 
acids.  It  is  hardly  possible  to  give  exact  figures  in  all 
cases,  because  those  found  by  the  different  authors  vary 
to  a  certain  degree,  and  because,  for  some  of  the  com- 
pounds, the  heats  of  combustion  have  not  been  measured. 
It  was  necessary  to  calculate  them  from  the  capacity  of 


THE    OXIDASES 


139 


oxygen,  and  it  is  possible  that  the  true  values  are  smaller 
or  greater  by  10  or  20  calories.  Nevertheless,  the  con- 
clusion cannot  be  denied  that  the  conversion  of  glucose 
or  stearic  acid  into  one  of  these  compounds  sets  free  a 
quantity  of  heat  which  is  not  sufficient  to  explain  the  heat 
really  produced  in  muscle  by  combustion  of  sugar  or  fat. 
Heat  of  combustion  of  100  gm.  glucose  and  the  corre- 
sponding quantities  of 

Glucose 380  Cal. 

Formic  acid 317 

Gly colic  acid 271 

Glyoxylic  acid.  .  .  .213 

Lactic  acid 329 

Ethyl  alcohol 357 

Formaldehyde.  .  .  .362 
Glycerinaldehyde.  .355 

Glyoxal 297 

Methylglyoxal  .  .  .  .338 


al.   380 

Cal. 

=  100 

per 

cent 

63 

"   17 

u 

109 

"   29 

a 

"    167 

"       44 

<< 

5i 

":  13 

a 

23 

6 

a 

18 

"   5 

<« 

25 

"   7 

(l 

83 

41   22 

it 

42 

"   11 

<■ 

Heat  of  combustion  of  100  gm.  stearic  acid  and  the 
corresponding  quantities  of 

Stearic  acid 975  Cal.  975  Cal.  =  100  per  cent. 

Acetaldehyde 877     "  98     "  "       10 

Oxybutyric  acid .  .745      "  230     "  "       24 

Acetone 660     "  315      "  "       32         " 

If  thirty  to  forty  per  cent  of  the  energy  developed  in  the 
muscle  becomes  work,  all  conversions  setting  free  less 
than  thirty  or  forty  per  cent  are  excluded  from  considera- 
tion in  seeking  an  explanation  of  the  source  of  energy  in 
muscle,  for  the  muscle  derives  no  energy  from  sub- 
stances which  are  subsequently  oxidized  elsewhere,  as  in 


140  ENZYMES 

the  blood  or  lungs.  Since  the  energy  is  needed  in  the 
muscles,  the  muscle-engine  naturally  cannot  use  energy 
set  free  elsewhere. 

Less  than  thirty  or  forty  per  cent  of  the  whole  potential 
energy  is  set  free  by  all  the  partial  combustions,  as  shown 
above,  except  in  the  case  of  acetone  arising  from  fat,  and 
the  glyoxylic  acid,  which  might  be  formed  by  partial 
combustion  of  sugar.  It  is  remarkable  that  of  all  the 
compounds  suggested  as  intermediate  products  of  com- 
bustion, only  these  two  are  found  under  special  conditions 
in  the  urine  or  in  the  body.  Nevertheless  I  think  that  it 
is  very  improbable  that  these  or  similar  products  are  the 
end-products  of  the  metabolism  in  muscle,  and  are  com- 
pletely oxidized  elsewhere.  For  in  that  case,  all  energy 
set  free  in  muscle  must  become  mechanical  work,  and 
the  muscle  would  have  a  ratio  of  efficiency  of  almost 
one  hundred  per  cent.  Because  this  result  is  very  im- 
probable, we  must  conclude  that  the  source  of  energy  for 
the  striped  skeletal  muscle  must  be  a  complete  oxidation 
of  sugar  or  fat.  The  large  amount  of  oxygen  absorbed 
in  glands,  according  to  Barcroft,  and  the  uniformity  of 
the  respiratory  quotient  in  glands  and  muscles,  supports 
this  idea  for  the  glands  as  well. 

The  conclusion  does  not  hold  good,  perhaps,  for  un- 
striped  or  smooth  muscles.  I  have  measured  the  output 
of  the  carbon  dioxide  of  the  muscles  of  the  small  intestine 
in  a  state  of  activity,  and  Kehrer  l  did  likewise  with  the 


1  E.  Kehrer:    Arch.  f.  Gynakologie,  89  (1909). 


THE    OXIDASES  141 

smooth  muscles  of  the  uterus.     The  production  of  carbon 
dioxide  per  ioo  gm.  per  hour  was  for 

Smooth  muscles       Smooth  muscles 
Striped  muscle  of  intestine  of  uterus  Kidney 

400  mgm.         79-90  mgm.         25    mgm.     1200  mgm. 

I  have  determined *  further  the  output  of  carbon 
dioxide  in  animals  with  striped  muscles,  such  as  insects, 
and  in  animals  with  unstriped  muscles,  such  as  snails 
and  earth-worms.  The  production  per  ioo  gm.  per  hour 
was  for 

Insects  Snails  Worms 

133-295  mgm.  n-16  mgm.  8-15  mgm. 

Magnus  2  has  observed  that  the  heart  beats  continued 
for  thirty  minutes  without  any  oxygen,  on  allowing  hydro- 
gen to  pass  through  the  vessels  of  the  heart.  The  absorp- 
tion of  oxygen  has  been  measured  in  the  heart  according 
to  Langendorff's  method  by  Barcroft,  who  found  it  to 
be  low.  It  seems  to  be  demonstrated  that  even  the 
central  nervous  system  of  frogs  can  live  with  but  a  very 
small  amount  of  oxygen,  if  the  poisonous  products  formed 
in  metabolism  are  eliminated  in  time.  The  difference 
between  striped  muscles  and  glands  on  the  one  hand  and 
the  smooth  muscles,  the  heart,  and  possibly  the  nervous 
system,  on  the  other,  is  so  striking,  that  the  two  are  per- 
haps governed  by  different  laws  of  metabolism.  Heart, 
smooth  muscles,  and  the  nervous  system  are  the  oldest 

1Zeitschr.  f.  physiol.  Chem.,  76  (1912). 

2  R.  Magnus:    Arch.  f.  exper.  Path.  u.  Pharmak.,  47  (1902). 


142  ENZYMES 

organs  in  phylogeny,  and  are  of  the  first  importance.  It 
may  be  that  cold-blooded  invertebrates  and  hibernating 
mammals  are  enabled  to  live  in  starvation  because  of  the 
small  amount  of  oxygen  needed  for  these  organs.  Animals 
with  smooth  muscles,  like  worms,  or  animals  without 
movement,  like  the  pupa  of  insects,  or  micro-organisms 
like  yeast  or  the  Bacillus  acidi  lactici,  can  live  with  an 
expenditure  of  only  a  small  amount  of  energy.  You  see 
that  worms,  yeast,  and  some  bacilli  do  indeed  convert 
sugar  without  oxidation.  They  can  live  with  an  efficiency 
of  the  body-engine  of  from  six  to  thirteen  per  cent. 

But  of  the  body  of  the  higher  animals  more  than  ninety 
per  cent  of  the  living  material  consists  of  striped  muscles 
and  glands,  and  their  metabolism  is  an  oxybiotic  one. 
The  enzymes  which  liberate  energy  in  the  tissues  of  the 
higher  animals  must  be  oxidases. 

After  this  digression  we  may  now  return  to  our  main 
subject. 

It  is  only  a  question  as  to  whether  the  oxidases  men- 
tioned before — aldehydase,  tyrosinase,  and  laccase — are 
the  true  metabolism-enzymes,  or  whether  they  have  other 
functions.  The  tyrosinase  and  the  laccase  convert  the 
aromatic  cleavage  products  of  the  proteins  into  compounds 
which  grow  gradually  darker,  and  become  immediately 
insoluble  in  the  fluids  of  the  animal  or  vegetable  organism. 
There  can  be  no  doubt  that  the  substances  thus  formed 
are  not  used  further  in  metabolism.  In  the  case  of  the 
lac  tree  or  the  hemolymph  of  insects,  they  have,  perhaps, 
a  protective  value.     The  tyrosinase  in  the  green  leaves  of 


THE    OXIDASES  143 

trees  produces  the  red,  yellow,  or  brown  colors  which 
are  so  ravishing  during  the  Indian  summer,  but  they  attack 
only  the  proteins  of  the  dead  or  dying  cells.  The  tyro- 
sinase in  the  ink-bag  of  the  sepia,  and  certainly  all  other 
oxidases,  have  an  important,  special  function,  but  do 
not  set  energy  free  for  utilization  in  the  processes  of  life. 
Whether  tyrosinases  occur  in  the  organism  of  higher 
animals,  is  not  determined.  In  human  pathology,  cases 
of  the  so-called  alkaptonuria  are  known.  In  such  cases, 
all  the  tyrosine  and  phenylalanine  of  the  food  is  converted 
into  homogentisic  acid: 

CH  COH 

CH^CH  HC^CH 


CH 


/CH  HCX 


CCH,COOH 


CHXHNH.COOH         COH 


This  resembles  the  conversion  of  tyrosine  by  tyrosinase, 
because  the  substances  formed  darken  in  the  presence  of 
the  oxygen  of  the  air.  The  urine  of  patients  with  alkap- 
tonuria darkens  in  a  few  minutes  or  hours,  and  thus 
the  abnormality  is  detected.  It  is  not  certain  whether 
homogentisic  acid  is  normally  formed  in  the  metabolism 
of  all  men,  and  the  further  conversion  of  the  homogentisic 
acid  checked  by  another  enzyme  or  group  of  enzymes,  or 
whether  the  normal  organism  burns  tyrosine  in  some  other 
way.  In  the  first  case,  we  would  have  to  conclude  that 
the  human  body  contains  an  enzyme  resembling  tyrosinase. 

The  aldehydase  of  the  liver  and  other  organs  may  be 
an  enzyme  destined  for  use    in  metabolism.     It  attacks 


144  ENZYMES 

not  only  salicylaldehyde,  but  other  aldehydes  as  well. 
The  sugars  are  aldehydes,  and  Neubauer  *  has  demon- 
strated that  phenylalanine,  and  perhaps  other  amino-acids, 
become  first  keto-  or  aldehydo-acids.  Thus  the  action 
of  aldehydase  is  perhaps  the  first  step  in  the  definite 
combustion  of  the  food.  Its  own  action  sets  free  no 
energy,  or  but  a  very  small  quantity,  but  it  prepares  the 
sugars,  etc.,  for  further  conversion.  It  is  possible  that 
aldehydase  has  special  functions,  not  known  to-day,  and 
that  we  may  have  to  separate  the  oxidases  thus  far  men- 
tioned from  the  metabolism-enzymes  in  a  strict  sense; 
like  zymase  and  the  glycolytic  enzyme  of  muscles. 

1  O.  Neubauer:    Deutsch.  Arch.  f.  klin.  Med.,  95  (1909). 


CHAPTER   XXI 

The  Metabolism-Enzymes 

The  metabolism-enzymes  in  the  strict  sense  bring  about 
the  combustion  of  food,  and  set  free  the  energy  needful 
for  life.  They  are  the  most  important  of  all  enzymes, 
because  digestion  and  all  other  conversions  are  only 
preparatory  for  the  real  combustion  of  food.  But  the 
investigation  of  these  enzymes  is  more  difficult  than  is  the 
investigation  of  the  enzymes  already  cited. 

That  combustion  in  protoplasm  is  the  work  of  enzymes 
has  been  for  a  long  time  assumed  by  many  investigators. 
But  more  recent  students  have  held  that  no  chemical 
substance  can  oxidize  sugar  or  fat  or  protein,  but  that- 
the  protoplasm-engine  with  its  peculiar  structure  can 
alone  carry  out  this  process.  The  question  was  answered 
by  Buchner's  discovery  of  zymase,  which  causes  the  con- 
version of  sugar  into  carbon  dioxide  and  alcohol,  and 
which  delivers  all  the  required  energy  for  the  life  of  the 
yeast.  Buchner  1  was  able  to  extract  from  two  other 
micro-organisms  similar  enzymes,  which  convert  sugar 
into  lactic  and  acetic  acid.  This  discovery  justified  the 
expectation  that  corresponding  enzymes  would  be  detected 
in  the  organs  and  tissues  of  animals. 

1  E.  Buchner  and  collaborators:  Ber.  d.  deutsch.  chem.  Ges.,  1896- 
1908. — E.  and  D.  Buchner  and  M.  Hahn:  "Die  Zymasegarung,"  Mu- 
nich, 190,3. 

10  145 


146  ENZYMES 

The  first  of  such  metabolism-enzymes  found  in  the 
tissues  of  higher  animals  is  one1  yielded  by  the  muscles 
of  dogs,  cats,  and  oxen.  This  enzyme  attacks  glucose 
and  changes  it  so  that  Fehling's  or  Pavy's  or  Trommer's 
test  no  longer  shows  the  presence  of  sugar.  The  exact 
chemical  process  has  not  as  yet  been  determined,  and  we 
have  no  decisive  proof  that  we  are  dealing  with  the  true 
combustion  of  glucose,  which,  as  already  mentioned, 
must  in  muscles  be  complete  in  order  to  furnish  the  re- 
quisite amount  of  energy.  We  meet  here  with  a  complica- 
tion not  occurring  in  the  unicellular  organisms.  Two 
substances,  from  two  different  organs,  must  unite  to  form 
the  enzyme  converting  the  sugar;  one  substance  originat- 
ing in  the  muscles  themselves,  the  other  produced  in  the 
pancreas,  and  reaching  the  muscles  only  when  needed. 
Further  evidence  for  the  enzymotic  nature  of  combus- 
tion in  metazoa  and  higher  animals  has  been  brought 
forward  by  recent  observations  of  Warburg.2  J.  Loeb 
has  suggested,  and  Warburg  has  supported  his  view,  that 
oxidation  in  cells  is  dependent  on  the  presence  of  the 
nucleus.  And  Warburg  has  observed  young,  red  cor- 
puscles in  human  and  rabbit's  blood,  which  show  a 
metabolism,  absorb  oxygen,  and  give  out  carbon  dioxide, 
but  possess  no  visible  nucleus.  They  are  newly  formed  in 
the  red  marrow  after  the  loss  of  large  quantities  of  blood, 
but  they  have    already  lost   the  nucleus,  and  cannot  be 

1  O.  Cohnheim:    Zeitschr.  f.  physiolog.  Chem.,  30,  42,  and  47  (1903- 
1906). 

2  O.  Warburg:     Zeitschr.  f.  physiolog.  Chem.,  57,  60  (1909). 


THE    METABOLISM-ENZYMES  147 

colored  with  alkaline  stains  which  show  affinity  for  the 
nucleus.  Nevertheless  these  corpuscles  yield  nucleic 
acid,  and  Warburg  1  and  Morawitz  2  were  able  to  observe 
relations  between  the  quantity  of  nucleic  acid  and  the 
amount  of  oxygen  absorption.  In  this  case  the  structure 
of  the  nucleus  is  lost,  but  the  absorption  of  oxygen  con- 
tinues, and  must,  therefore,  be  provoked,  not  by  any 
structural  engine,  but  by  a  chemical  substance,  that  is  to 
say,  by  an  enzyme. 

Further  evidence,  though  not  so  conclusive,  is  given  by 
the  experiments  of  Vernon,3  in  which  absorption  of 
oxygen  and  production  of  carbon  dioxide  continued  for 
a  long  time,  when  he  placed  surviving  kidneys  and  other 
organs  in  well-oxygenated  Ringer's  solution.  It  appears 
that  cells  perish  early  and  suddenly,  and  at  the  moment 
of  dying  the  amount  of  gaseous  metabolism  diminishes, 
but  continues  to  a  certain  extent,  and,  even  during  this 
time,  the  tissues  absorb  oxygen  and  yield  carbonic  acid 
in  the  same  ratio  as  before.  According  to  Vernon,  this 
signifies  that  real  respiratory  substances  exist  which 
occasion  combustion  under  these  as  well  as  normal  con- 
ditions. 

All  the  foregoing  considerations  lead  to  the  expectation 
that  the  metabolism  of  higher  animals  will  be  proved  to 
be  due  to  enzymes,  and  that  only  the  technical  difficulties 
of  investigation  have  thus  far  prevented  us  from  discover- 

1  O.  Warburg:    Zeitschr.  f.  physiolog.  Chem.,  59  (1909). 

2  P.  Morawitz:   Arch.  f.  exper.  Path.  u.  Pharm.,  60  (1909). 

3  H.  M.  Vernon:    Journ.  of  Physiology,  35,  36,  and  39  (1906-1909). 


148  ENZYMES 

ing  more  of  the  enzymes  participating  in  the  process. 
That  enzymes  should  be  the  cause  of  all  combustion  in 
the  animal  body,  is  a  matter  of  great  importance.  There 
can  be  no  doubt  that  the  production  of  enzymes,  and  the 
control  of  their  action,  depend  upon  the  living  protoplasm. 
By  the  discovery  of  the  metabolism-enzymes,  one  stage 
of  the  living  process  becomes  a  simple  chemical  process, 
and  we  are  enabled  to  hope  that  further  investigations 
will  show  that  subsequent  stages  are  susceptible  of  equally 
simple  explanation. 

We  are,  to-day,  far  from  attaining  this  goal,  but  we  can 
show  that  the  surviving  organ  with  its  structure  acts  in 
another  way  than  any  extract  of  the  same  organ.  For 
instance,  extracts  of  the  mucous  membrane  of  the  small 
intestine  of  fish  contain  erepsin;  the  extracts  dissociate 
peptone  into  amino-acids,  but  leave  these  amino-acids 
untouched.  But  I  have  placed  the  surviving  intestine 
of  these  same  fish  (Labrus  or  Crenilabrus)  in  Ringer's 
solution,  filled  the  intestine  with  a  solution  of  peptone  or 
tyrosine,  and  allowed  the  intestine  floating  in  the  Ringer's 
solution  to  absorb  the  peptone  solution  and  thus  to  transfer 
its  contents  into  the  Ringer's  solution  outside.  In  a  high 
invertebrate,  the  Octopus,  I  found  amino-acids,  but  in 
fish,  during  the  passage  through  the  wall,  peptone  and 
tyrosine  are  transformed.  Ammonia  is  split  off  in  great 
amount.1 

A  second  example  is  the  liver.     If  we  allow  liver  extract 

1  O.  Cohnheim:    Zeitschr.  f.  physiolog.  Chem.,  59  (1909). 


THE   METABOLISM-ENZYMES 


149 


to  act  upon  amino-acids,  they  are  not  at  all  changed;  but 
if  we  use  the  undestroyed  liver  as  a  surviving  organ,  and 
cause  a  stream  of  blood  to  flow  through  the  vessels,  then 
leucine,  isovaleric  acid,  and  some  other  cleavage-products 
of  proteins,  when  added  to  the  blood,  are  converted  into 
acetone,  as  has  been  pointed  out  by  Embden.1 


CH3     CH3 

\  / 

CO 

Acetone 


CH3     CH3 

\   / 

CH 

Leucine    CH2         — * 
CNH2 

COOH 

CH3  CH3 

\  / 

CH    - 

CH2 

COOH 

Isovaleric  acid 

A  third  less  evident  example  which  can  be  cited  against 
the  theory  of  oxidation  accomplished  by  enzymes,  is  the 
absorption  of  oxygen  by  the  eggs  of  the  sea-urchin,  studied 
by  Warburg.2  The  oxidation  is  influenced  by  the  sur- 
face of  the  eggs. 

The  difficulties  of  obtaining  and  bringing  zymase  into 


CH3    CHa 


CO 
Acetone 


1  G.  Embden:    Hofmeister's  Beitr.,  8  (1906). 

2  O.  Warburg:    Zeitschr.  f.  physiolog.  Chem.,  66  (1910). 


150  ENZYMES 

solution  was  mentioned  in  the  early  lectures.  Zymase  can 
be  precipitated  together  with  the  proteins  of  the  press- 
juice,  by  acetone  or  by  alcohol  and  ether,  or  it  can  be 
salted  out  like  other  enzymes.  It  is  resistant  to  changes 
in  reaction  like  living  yeast,  but  suffers  destruction  in  a 
short  time  whatever  the  reaction.  It  is  more  stable  when 
allowed  to  act  in  the  presence  of  sugar  than  in  the  isolated 
state,  but  even  then  loses  activity  within  three  to  four 
days.  Yeast,  and  therefore  the  press-juice  of  yeast, 
yields  a  proteolytic  enzyme,  the  so-called  endotrypsin  or 
endotryptase.  Buchner  has  not  succeed  in  separating 
this  enzyme,  and  suggests  that  zymase  is  destroyed 
by  endotryptase,  and  that  this  is  the  reason  for  its  in- 
stability. The  action  of  the  zymase  is  increased  if  we 
add  sodium  phosphate  to  the  press-juice.  Boiled  press- 
juice  or  extracts  of  boiled  yeast,  which  contain  phosphates, 
likewise  augment  the  action  of  zymase.  It  is  not  certain 
whether  phosphates  improve  the  action  of  the  zymase  by 
influencing  the  reaction  or  other  conditions,  or  whether  it 
functionates  as  a  true  co-ferment,  like  the  manganese  in 
the  case  of  laccase,  or  the  glycocholic  and  taurocholic 
acids  in  the  case  of  pancreatic  steapsin. 

The  qualities  of  the  lactacidase  and  the  alcohol- 
oxidase  yielded  by  the  bacteria  that  convert  sugar  into 
lactic  acid,  or  alcohol  into  acetic  acid,  which  have  been 
studied  by  Buchner  and  Herzog,1  resemble  the  qualities 
of  zymase  so  far  as  difficulty  of  extraction  and  instability 

1  See  J.  Meisenheimer:    Biochem.  Zentralbl.,  6  (1907). 


THE   METABOLISM-ENZYMES  151 

are  concerned.  Plants  of  different  classes  contain,  accord- 
ing to  Hahn,1  Palladin,3  Stoklasa,3  and  other  authors, 
enzymes  which  convert  sugar,  and  these  are  called  respira- 
tory enzymes.  Most  of  them  form  alcohol  like  zymase, 
but  it  seems  that  alcohol  is  not  always  formed,  and  then 
only  when  oxygen  is  lacking.  The  chemical  processes  in 
plants  in  the  presence  of  oxygen  are  less  understood. 
The  old  claims  that  yeast  also  ferments  sugar  only  when 
there  is  a  lack  of  oxygen,  have  been  refuted  by  Buchner. 
The  glycolytic  enzyme  in  the  animal  body  seems  to  be 
even  more  unstable  than  the  metabolism-enzymes  of 
bacteria.  Stoklasa  thought  he  had  found  enzymes  in 
the  animal  tissues  which  could  convert  sugar  and  form 
alcohol.  There  can  be  no  doubt  that  he  was  deceived 
by  experimental  error.  He  did  not  observe  tissue- 
enzymes,  but  bacteria  grew  in  his  solutions,  which  were 
not  protected  by  antiseptic  substances  in  sufficient  quan- 
tity, and  they  developed  carbon  dioxide  and  formed 
alcohol.  If  we  add  glucose  to  muscle  extract  prepared  by 
any  process,  no  sugar  is  changed;  but  the  sugar  is  attacked, 
as  I  have  already  stated,  if  we  add  to  the  muscle  extract  a 
certain  substance — the  co-ferment  of  the  glycolytic  enzyme 
of  muscle,  yielded  by  the  pancreas.  This  glycolytic 
enzyme  is  highly  sensitive  to  an  acid  reaction,  and  is 
destroyed,  even  in  the  frozen  state,  in  one  or  two  days,  and 
in  a  few  hours  at  the  temperature  of  the  room  or  body. 

1  M.  Hahn:    Ber.  d.  deutsch.  chem.  Ges.,  33  (1900). 

2  W.  Palladin:    Zeitschr.  f.  physiolog.  Chem.,  55,  56  (1908). 

3  J.  Stoklasa:    ibid,,  50  (1906). 


ALU . 

COLLEGE  Of 


152  ENZYMES 

Because  the  muscle  either  does  not  contain  a  proteolytic 
enzyme,  or  contains  a  very  weak  one,  the  loss  of  power 
cannot  be  due  to  a  destruction  by  a  trypsin-like  enzyme. 
The  presence  of  oxygen  is  necessary  or  at  least  has  a 
favorable  influence  upon  the  action  of  the  glycolytic 
enzyme. 

Vernon  x  has  tried  to  throw  light  upon  the  chemical 
character  of  respiratory  substances,  that  is,  of  enzymes, 
and  he  observed  the  gaseous  exchange  of  surviving  organs 
after  the  addition  of  poisons.  He  saw  that  hydrocyanic 
acid,  some  metals,  hydroxylamin,  and  other  substances 
which  react  easily  with  aldehydes,  are  poisons  which  check 
oxygen  absorption,  and  he  suggests  that  perhaps  respira- 
tory substances  resemble  aldehydes. 

No  other  metabolism-enzymes  have  thus  far  been 
isolated  from  the  tissues  and  cells,  though  it  is  a  probable 
hypothesis  that  enzymes  take  part  in  all  the  chemical 
processes  of  the  body.  But  if  we  accept  this  hypothetical 
view,  we  must  assume  the  existence  of  the  following 
metabolism-enzymes,  though  they  have  not  as  yet  been 
found  and  only  the  actions  to  which  they  give  rise  are 
known.  With  a  mental  reservation,  we  may  assume  their 
existence  from  their  actions : 

i.  Oxidases  which  completely  burn  fats,  sugar,  and 
proteins  in  the  muscles  and  glands  of  the  higher  animals. 

I  have  already  stated  that  anoxybiotic  processes,  mere 
splittings,  are  incompatible  with  the  active  processes  of 

1  H.  M.  Vernon:    Journal  of  Physiology,  39  (1909). 


THE    METABOLISM-ENZYMES  153 

animal  metabolism,  and  that  fats  and  sugar  must  be 
completely  oxidized.  Nevertheless  it  is  possible  that 
oxidation  is  accomplished  by  several  enzymes,  working 
one  after  another,  like  the  proteolytic  enzymes  of  the 
alimentary  canal.  Buchner  has  suggested  that  zymase 
does  not  immediately  form  alcohol  and  carbon  dioxide, 
but  first  forms  lactic  acid,  and  the  lactic  acid  is  then  con- 
verted into  alcohol  and  carbon  dioxide  by  a  second, 
separate  enzyme.  The  proofs  brought  forward  for  this 
view  do  not  seem  to  me  to  be  conclusive.  The  suggestion 
is  only  supported  by  the  observation  of  very  small  quanti- 
ties of  lactic  acid  besides  the  alcohol,  and  by  the  fact  that 
this  small  quantity  of  lactic  acid  varies  with  individual 
yeasts.  But  lactic  acid  can  also  be  formed  from  other 
products  produced  by  the  yeast,  especially  from  alanine 
and  protoplasmic  proteins,  and  set  free  by  the  endo- 
tryptase  occurring  in  all  yeasts  and  in  all  preparations  of 
zymase.  In  higher  animals,  Zuntz  and  his  collaborators 
have  observed  that  the  absorption  of  oxygen  and  the  out- 
put of  carbon  dioxide  increases  immediately  when  the 
work  done  by  the  muscles  or  glands  increases,  and  that 
the  respiratory  quotient,  the  proportion  C02/02,  is  not 
at  all  changed  if  work  is  done  by  muscles.  Neither  is 
oxygen  stored  up  even  for  a  few  minutes,  nor  is  carbon 
retained  anywhere  in  the  organism.  If  we  are  really 
dealing  with  a  plurality  of  enzymes,  these  enzymes  must 
work  simultaneously;  they  cannot  be  separated  in  space 
or  time. 

2.  Enzymes  which  split  fats  and  amino-acids,  and  per- 


154  ENZYMES 

haps  sugars,  at  different  stages,  and  thus  form  intermediate 
products  which  may  have  special  functions  in  the  organism, 
or  may  be  used  for  building  new  material,  or  they  may 
occur  under  abnormal  conditions,  when  an  enzyme  or  a 
function  fails.  We  know  now  the  following  cases,  but  I 
wish  to  emphasize  that  the  enzymotic  nature  of  these 
processes  is  to-day  a  mere  hypothesis.  Experimental  ob- 
servations tell  us,  on  the  contrary,  that  all  these  processes 
are  connected  with  the  structure  of  the  organs.  They 
cannot  be  observed  in  extracts,  but  they  can  be  observed 
only  in  the  living  organism  or  surviving  organs. 

i.  Butyric  acid  is  converted  into  ^-oxybutyric  acid  in 
diabetes. 

2.  Fatty  acids,  as  palmitic  and  stearic  acids,  are  con- 
verted into  /?-oxybutyric  acid  and  acetoacetic  acid.1  The 
conversion  occurs  in  human  diabetes,  pancreatic  diabetes, 
and  in  starvation,  i.e.,  when  no  sugar  is  burned.  It 
occurs  in  the  surviving  liver.  The  conversion  of  B- 
oxybutyric  acid  into  acetoacetic  acid  is  a  reversible  process 
(see  page  133). 

3.  Some  cleavage-products  of  proteins,  leucine,  tyrosine, 
phenylalanine,  phenyl-lactic  acid,  homogentisic  acid, 
isovaleric  acid,  as  well  as  butyric  and  oxybutyric  acids, 
are  converted  into  acetone.  The  conversion  occurs  in  the 
liver.2 

1  H.  C.  Geelmuyden:  Skandinav.  Arch.  f.  Physiol.,  n  (1900). — A. 
Magnus-Levy:  Arch.  f.  exper.  Path.  u.  Pharmak.,  42  (1899);  45  (1901). 
— J.  Baer  and  L.  Blum:  ibid.,  55,  56  (1906). — Hofmeister's  Beitr.,  1 
(1907). 

2  G.  Embden:    ibid.,  8  (1906). — L.  Borchardt:    ibid.,  9  (1907). 


THE   METABOLISM-ENZYMES  155 

4.  Glycerin  is  converted  into  glucose  or  glycogen.1  The 
conversion  occurs  in  diabetes. 

5.  Alanine,  glutamic  acid,  and  perhaps  other  cleavage- 
products  of  protein,  are  converted  into  sugar.  The  con- 
version occurs  in  diabetes.2 

6.  Glucose  is  converted  into  glycuronic  acid : 


CH2OH 

COOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

COH 

COH 

The  conversion  occurs  when  such  substances  like 
chloral,  thymol,  camphor,  etc.,  enter  the  organism.  They 
are  conjugated  with  glycuronic  acid  in  the  liver. 

7.  Leucine,  isoleucine,  and  valine,  are,  according  to 
Felix  Ehrlich,3  converted  into  isoamyl  alcohol,  active 
isoamyl  alcohol,  and  isobutyl  alcohol. 

The  three  alcohols  form  the  fusel  oil  found  in  liquors 
manufactured  by  yeast-fermentation.  Corresponding  en- 
zymes convert  other  cleavage-products  of  proteins  into 
alcohols,  which,  coupled  with  other  alcohols,  form  the 
odorous  compounds  of  roses,  apples,  pears,  and  other 
flowers  and  fruits. 

1  M.  Cremer:  Miinchener  med.  Woch.,  1901. — H.  Liithje:  Deutsch. 
Arch.  f.  klin.  Med.,  80  (1904). 

2  G.  Lusk:   American  Journ.   of  Physiol.,  25,  1909. 

3  F.  Ehrlich:  Biochem.  Zeitschr.,  2  (1906). — H.  Thierf elder:  Zeitschr. 
f.  physiol.  Chem.,  11  (1887). 


156 


ENZYMES 

CH3    CH3 
\  / 
CH 

CH3       CH3 
\CH2 

CH2 
CHNH2 

(Leucine) 

CH           (isoleucine) 
CHNH2 

COOH 

l 

COOH 

i 

CH3    CH3 

\  / 

CH           (Amyl  alcohol) 

CH2 

CH2OH 

1 
CH3       CH3 

\  /  CH2            (Active 
CH       isoamyl  alcohol) 
CH2OH 

CH3     CH3 

\  / 

CH 

CHNH2 

(Valine) 

COOH 

i 

1 
CH3     CH3 

\  / 

CH 

(isobutyl  alcohol) 

CH2OH 

8.  Amino-acids  split  off  ammonia  in  passing  through 
the  intestinal  wall  of  fish.1 

These  aminolytic  enzymes  seem  to  be  widely  distributed 
in  both  the  vegetable  and  animal  kingdoms,  as  well  as 
in  bacteria.  The  well-known  products  of  putrefaction, 
skatol,  phenylacetic  acid,  etc.,  are  formed  by  aminolytic 
processes.  These  aminolytic  enzymes  do  not  set  free 
energy,  and  it  might  be  easier  to  isolate  them  than  the 
metabolism-enzymes  in  the  strict  sense. 


1  O.  Cohnheim:    Zeitschr.  f.  physiol.  Chem.,  59,  61  (1909). 


THE   METABOLISM-ENZYMES  157 

I 

9.  Ornithine  and  lysine  are  converted  into  putrescine 
and  cadaverine. 


CO. 


CH2NH2 

CH2NH2 

CH2 

CH2 

CH2 

CH2         + 

CHNH2 

CH2NH2 

COOH 

The  conversion  occurs  through  the  action  of  putrefac- 
tion bacteria,  and  in  human  metabolism  in  cases  of 
cystinuria. 

10.  Cysteine  is  converted  into  taurine. 


CH2SH  CH2S02OH 

CHNH3  +  30=  CH2NH2        +  COa 

COOH 

This  conversion  occurs  in  the  liver.1 
11.  Tyrosine    and    phenylalanine    are  converted    into 
homogentisic  acid  in  the  so-called  alkaptonuria.2,3 

COH  CH 

HC    CH  HC    CH 

HC     CH  HC     CH 

CCH2CHNH2COOH  CCH2CHNH2COOH 

Tyrosine  Phenylalanine 

1  G.  v.  Bergmann:    Hofmeister's  Beitr.,  4  (1904). 

2E.  Baumann  and  v.  Udransky:    Zeitschr.  f.  physiolog.  Chem.,  13,  15 
(1889). — A.  Ellinger:    ibid.,  29  (1900). 

3  E.  Meyer:    Deutsch.  Arch.  f.  klin.  Med.,  70  (1901).' 


158  ENZYMES 

CH 

/\ 

HC    COH 

II      I 
HOC     CH 


CCH2COOH 

Homogentisic  acid 

12.  Lactic  acid  is  formed  from  an  unknown  compound 
in  muscles.  An  enzyme  widely  distributed  in  the  organ- 
ism causes  the  acid  reaction  in  tissues  and  organs  im- 
mediately after  death,  or  under  special  conditions  during 
life.  The  reaction  1  of  the  blood,  lymph,  and  tissues  of 
animals,  is  neutral,  and  the  organism  is  well  provided  with 
means  to  maintain  the  neutrality  of  its  tissues.  It  is 
maintained  (i)  by  the  twofold  character  of  carbonic 
acid,  which  can  react  as  an  acid  with  sodium  and  neutralize 
it,  or  can  become  C02,  a  neutral  gas;  (2)  by  the  twofold 
character  of  proteins  and  all  cleavage  products  of  proteins, 
which  are  amphoteric  electrolytes,  and  react  both  with 
bases  and  acids,  and  neutralize  them  to  a  certain  extent; 
(3)  by  the  presence  of  phosphates  on  the  one  hand,  and 
of  ammonia  on  the  other;  and  (4)  by  burning  all  acids 
formed  in  the  immediate  metabolism  into  neutral  carbon 
dioxide.  But  this  combustion  is  an  oxidation,  and  where 
oxygen  is  lacking,  acid  reaction  occurs.  Thus  it  occurs 
immediately  after  death.  In  warm-blooded  animals, 
especially  the  larger,  the  high  temperature  of  the  body 

1  O.  Cohnheim:  "Physiologie  der  Verdauung  u.  Ernahrung,"  Berlin, 
p.  347.     L.  Henderson:    Ergebnisse  der  Physiologie,  8  (1909). 


THE    METABOLISM-ENZYMES  159 

remains  for  a  time  after  death,  and  the  enzymes  can  then 
still  act  energetically.  But  oxidation  is  checked  with  the 
last  heart-beat,  and  the  tissues  now  become  acid  in  re- 
action. We  can  observe  this  acid  reaction  in  the  liver, 
the  pancreas,  the  kidney,  the  brain,  and  the  muscles.  If 
we  kill  dogs,  cats,  or  rabbits  in  the  laboratory,  and  study 
the  organs  a  few  minutes  after  death,  we  find  that  the 
reaction  of  the  kidney  and  the  brain  may  be  acid,  because 
these  organs  have  the  greatest  metabolism,  while  the  liver 
and  the  pancreas  may  still  exhibit  a  neutral  reaction. 
From  the  ox  we  can  obtain  the  liver  and  pancreas  only 
forty  to  sixty  minutes  after  death,  and  in  this  time  these 
glands  become  acid. 

In  muscles,  an  acid  reaction  produces  rigor  mortis. 
Kiihne  was  the  first  to  show  that  rigor  mortis  is  the 
clotting  of  the  fluid  muscle-plasma,  and  that  a  change  in 
the  myosin  is  the  cause  of  this  clotting.  Many  authors 
have  supposed  that  myosin  is  coagulated  by  a  ferment,  like 
fibrinogen  by  thrombin,  or,  as  a  common  example,  like 
casein  by  rennet.  But  the  occurrence  of  rigor  mortis 
is  always  connected  with  an  acid  reaction;  myosin  is 
precipitated  like  many  other  proteins,  as  the  so-called 
globulins,  by  acid.  By  the  precipitation,  the  fluid  sarco- 
plasm  becomes  solid  in  the  muscle-fibrils,  and  rigor  mortis 
occurs.  Later,  the  solid  coagulum  contracts,  and  squeezes 
out  the  fluid,  just  as  the  blood  serum  is  squeezed  from 
clotted  blood  and  the  rigor  mortis  is  relaxed  again.  Thus 
the  formation  of  acid  in  muscles  is  an  important  phenome- 
non, and  investigators  have  tried  to  throw  light  upon  the 


160  ENZYMES 

process.  The  acid  formed  in  muscle  deprived  of  arterial- 
ized  blood  is  lactic  acid,  and  the  formation  of  lactic  acid 
under  different  conditions,  the  time  of  formation,  the  quan- 
tity of  formation,  etc.,  have  been  pointed  out  recently 
by  Fletcher  and  Hopkins.1  The  chief  result  obtained  by 
these  authors  is  that  the  formation  of  lactic  acid  is  closely 
connected  with  the  life  of  the  muscles,  but  only  with  life 
under  abnormal  conditions,  especially  with  lack  of  oxygen. 
If  sufficient  oxygen  is  supplied,  not  only  is  no  lactic  acid 
formed,  but  even  lactic  acid  which  has  previously  developed 
is  destroyed  in  the  presence  of  oxygen.  It  might  be 
suggested  that  a  ferment,  occurring  in  muscles,  converts 
sugar,  with  a  free  supply  of  oxygen,  into  carbon  dioxide; 
and  with  a  lack  of  oxygen,  into  lactic  acid.  But  this  ex- 
planation is  not  in  agreement  with  the  results  of  Fletcher 
and  Hopkins,  that  lactic  acid  is  neither  formed  in  dead 
muscles  nor  in  muscle-extracts,  but  only  in  surviving 
muscles  deprived  of  oxygen,  and  they  emphasize  that  no 
evidence  has  been  brought  forward  for  sugar  as  the  source 
of  lactic  acid.  The  formation  of  lactic  acid  does  not  occur 
in  solution,  and  we  meet  here  again  with  the  phenomenon, 
that  some  reactions  are  limited  to  certain  structures,  and 
we  hypothesize  when  we  speak  of  an  enzyme  which  gives 
rise  to  lactic  acid  when  there  is  a  lack  of  oxygen. 

Lack  of  oxygen  occurs  after  death,  but  it  occurs  also 
during  life.  Araki,2  in  Hoppe-Seyler's  laboratory,  de- 
monstrated that  with  a  lack  of  oxygen,  dogs  and  rabbits 

1  W.  M.  Fletcher  and  F.  G.  Hopkins:    Journ.  of  Physiology,  35  (1907). 

2  T.  Araki:    Zeitschr.  f.  physiolog.  Chem.,  15  (1891). 


THE    METABOLISM-ENZYMES  161 

form  and  eliminate  great  quantities  of  lactic  acid,  and  it 
is  an  old  experiment,  that  the  contracting  frog's  muscles 
become  acid.  If  we  inject  litmus  solution  into  a  frog,  cut 
one  sciatic  nerve  and  stimulate  it,  the  stimulated  leg 
becomes  red,  while  the  unstimulated  leg  shows  a  blue 
or  violet  color.  It  is  necessary,  however,  to  tie  the  leg, 
and  prevent  a  free  oxygen  supply.  According  to  Zuntz,1 
acids,  probably  lactic  acid,  are  formed  in  hard  muscular 
work  which  exceeds  physiological  limits. 

The  formation  of  lactic  acid  is  one  of  the  reasons  which 
have  provoked  the  suggestion  of  the  anoxybiotic  processes 
in  tissues.  I  conclude  only,  that  tissues  yield  an  enzyme 
which,  under  special  conditions,  can  form  lactic  acid. 
The  lactic  acid  is  the  S-a-oxypropionic  acid,  corresponding 
to  alanine. 

1  N.  Zuntz  and  J.  Geppert:  Pfluger's  Arch.,  62  (1896).— A.  Bornstein 
and  E.  Poler:    ibid.,  95  (1903). 

11 


CHAPTER  XXII 

The  Fibrin-Ferments 

It  remains  but  to  mention  an  enzyme  which  differs  from 
all  enzymes  described  so  far,  the  fibrin-ferment,  which 
provokes  the  clotting  of  blood.  In  this  clotting  of  the 
blood  the  chemical  steps  are  known,  but  it  was  uncer- 
tain whether  an  enzyme  provokes  them.  The  enzymotic 
character  of  blood-clotting  cannot  be  denied,  and  the 
chemical  process  is  not  explained. 

Blood,  when  shed  from  the  blood-vessels  of  a  living 
body,  is  perfectly  fluid.  In  a  short  time,  however,  it 
becomes  viscid.  The  viscidity  increases  rapidly  until 
the  whole  mass  of  blood  becomes  a  complete  jelly.  The 
factors  playing  a  role  in  the  coagulation  of  blood  are 
extremely  complicated,  there  being  four  different  sub- 
stances which  react  upon  one  another.  In  the  first  place, 
the  blood  contains  the  soluble  fibrinogen,  which,  as  Ham- 
marsten  1  has  demonstrated,  occurs  in  the  circulating 
blood  as  a  special  albuminous  body.  Then  there  is 
the  true  fibrin-ferment,  which,  in  all  probability,  is  also 
present  in  circulating  blood  as  zymogen.2  The  third 
substance  is  the  activator  2  of  the  fibrin-ferment,  or  the 
thrombin,  as  it  is  called  after  activation. 

10.  Hammarsten:  Zeitschr.  f.  physiolog.  Chem.,  22  (1896);  30 
(1899). 

2 P.  Morawitz:  Hofmeister's  Beitr.,  4  (1903);  Deutsch.  Arch, 
f.  klin.  Med.,  79  (1903);  Ergebnisse  d.  Physiologie,  4  (1905). 

162 


THE    FIBRIN-FERMENTS  163 

The  activator  has  been  designated  as  "thrombo kinase." 
In  order  to  effect  the  change  induced  by  thrombo  kinase, 
a  fourth  factor,  namely  lime  salts,  is  necessary. 

The  process  by  which  fibrinogen  is  changed  into  fibrin 
is  not  known.  All  that  is  known  is,  that  an  insoluble 
substance  is  produced  which  has  always  been  considered 
as  being  similar  to  casein  which  clots  by  the  agency  of 
rennet.  But  whether  this  process  involves  a  cleavage, 
and  whether  one  of  the  products  of  such  cleavage  is 
the  insoluble  substance  referred  to,  is  a  question  we  are 
not  prepared  to  answer.  Nor  can  the  relations  be  ex- 
plained between  the  thrombin,  or  its  zymogen,  the  lime 
salts,  and  thrombokinase,  although  it  would  seem  that 
there  exists  a  certain  quantitative  relation  between  the 
kinase  and  the  thrombin  similar  to  that  of  the  activating 
process  of  trypsin  in  the  cases  already  referred  to.  Lime 
salts,  thrombin,  and  fibrinogen  are  present  in  the  blood. 
The  reason  why  circulating  blood  does  not  coagulate  is 
that  thrombokinase  is  absent.  Thrombokinase,  however, 
is  present  in  the  blood  platelets,  and  the  investigations  of 
Deetjen 1  have  rendered  it  probable  that  it  exists  no- 
where else.  The  blood  platelets  which  constitute  the 
third  component  of  the  blood — the  other  two  being  the 
red  and  white  blood  corpuscles — are  complete  cells,  just 
like  the  white  corpuscles,  provided  with  nucleus,  proto- 
plasm, and  ferments.  The  fact  that  formerly  so  little 
was  known  of  them  is  explained,  first,  by  the  fact  that 

1H.  Deetjen*  virchow's  Arch.,  164;  Zeitschr.  f.  physiolog.  Chem., 
63  (1909). 


164  ENZYMES 

they  are  smaller  than  the  leucocytes  and  red  blood  cor- 
puscles, and  second,  that  they  are  very  easily  destroyed 
and  go  into  solution.  Deetjen  was  the  first  to  succeed  in 
preserving  the  blood  platelets  outside  the  organism 
sufficiently  long  to  examine  thoroughly  and  fix  them. 
He  obtained  cultures  on  agar-agar  in  the  same  way  as 
bacteria  are  grown  on  the  same  substance;  and,  by 
adopting  special  precautions,  he  was  able  to  observe 
them  under  the  microscope. 

The  fact  that  these  blood  platelets  are  exceedingly 
sensitive  to  all  kinds  of  influences,  is  intelligible  from  the 
function  they  have  to  perform,  which  is  to  make  the 
blood  coagulate.  If  the  object  of  the  blood  coagulation, 
that  is,  the  inhibition  of  hemorrhage,  is  to  be  attained, 
it  is  necessary  for  the  blood  to  coagulate  whenever  it 
leaves  the  vessels.  Therefore,  the  mere  fact  of  blood 
flowing  out  under  abnormal  conditions  must  suffice  to 
coagulate  it,  and  for  this  reason  the  blood  platelets, 
which  contain  the  thrombokinase,  are  so  exceedingly 
sensitive  to  all  influences  which  are  exerted  upon  the 
blood  from  without  the  normal  vessels. 

Deetjen  has  found  two  facts:  (i)  The  blood  plate- 
lets perish  whenever  they  come  in  contact  with  a  rough 
surface;  and  it  is  a  well-established  fact  that  blood  coagu- 
lates not  only  outside  the  blood-vessels,  but  also  within 
them  whenever  the  normal  smooth  endothelium  has 
been  destroyed;  and  (2)  the  blood  platelets  invariably 
perish  when  the  blood  becomes  alkaline  in  reaction. 
Fluids   and   tissues   of   the   organism   under   physiologic 


THE   FIBRIN-FERMENTS  165 

conditions  always  have  a  rather  constant  neutral  reaction, 
and  the  organism  has  means  to  restore  this  reaction  if 
any  extraneous  influence  impairs  it.  (See  page  158.) 
We  have  mentioned  agents  which  prevent  its  turning 
acid.  In  the  present  instance,  we  have  to  consider 
agents  which  prevent  its  turning  alkaline,  because  the 
blood  platelets  are  destroyed  by  an  alkaline  reaction. 
Now,  alkaline  reaction  may  occur  under  two  conditions, 
one  of  which  is  an  artificial  one.  The  glass  utensils  used 
for  scientific  experiments  are  not  entirely  insoluble  in 
water;  after  a  while  they  communicate  to  the  water  some 
alkalinity,  and  these  small  quantities  of  alkali,  which 
exist  also  in  distilled  water  stored  in  glass  vessels,  even 
in  beakers,  slides,  and  cover  glasses,  are  sufficient  to 
destroy  the  blood  platelets.  Deetjen  has  found  that  it  is 
possible  to  observe  the  blood  platelets  properly  only  by 
discarding  glass  in  the  apparatus  used,  such  as  vessels, 
beakers,  test  tubes,  slides,  and  cover  glasses,  and  re- 
placing it  by  quartz,  which  does  not  communicate  alkali 
to  water. 

Furthermore,  carbonic  acid  is  contained  in  the  blood, 
both  as  gas  and  combined  with  sodium.  If  blood  comes 
into  contact  with  the  outside  air,  that  part  of  the  carbonic 
acid  which  is  present  as  gas  escapes;  and  the  relation 
between  the  carbonic  acid  and  the  sodium  is  loosened 
by  reason  of  the  reduced  carbonic  acid;  and  if  blood  has 
been  in  contact  with  the  extraneous  air  only  for  a  short 
time,  its  reaction  becomes  alkaline.  Even  this  slight 
alkalinity  is  sufficient  to  destroy  the  blood  platelets,  to 


166  ENZYMES 

permit  the  occurrence  of  thrombokinase,  and  to  cause 
coagulation  of  the  blood. 

The  relations  between  thrombokinase  and  the  pro- 
tease which  is  found  in  the  blood  platelets  (see  p.  112) 
are  not  understood.  According  to  Hammarsten,  the 
conversion  of  fibrinogen  into  fibrin  is  a  slight  dissociation 
and  it  can  be  possible  that  the  clotting  of  blood  is  a  pro- 
teolytic process  like  the  clotting  of  milk,  and  that  the 
weak  proteolytic  enzyme  of  blood  platelets  is  set  free  by 
this  dissolution  and  acts  as  fibrin-ferment. 

Fibrin-ferment  together  with  blood  coagulation  is 
therefore  one  of  the  means  of  protection  which  the  or- 
ganism employs  to  ward  off  hostile  interference,  and,  in 
connection  herewith,  we  come  to  a  special  class  of  fer- 
ments. Fibrin-ferment  has  nothing  to  do  with  metabolism 
in  the  normal  processes  of  the  organism;  it  is  merely  a 
factor  of  safety  with  which  the  organism  is  provided.  It 
will  thus  be  seen  in  what  effective  manner  the  properties 
of  the  blood  platelets  have  been  made  serviceable  to 
insure  the  safety  of  the  organism. 


INDEX    OF  AUTHORS 


Abelous,  126 
Abderhalden,  49,  52,  60, 

101,  in,  115,  117 
Araki,  160 
Armstrong,  40 
At  water,  136 

Bach,  125,  132 
Baer,  154 

Bainbridge,  78 

Bang,  119 

Barcroft,  136 

Barker,  113 

Bauman,  157 

Bayer,  90 

Bayliss,  1,  17,  21,  69 

Benedict,  136 

v.  Bergmann,  157 

Bergmann,  P.,  8,  90 

Bertrand,  71,  127 

Biarnes,  126 

Biernacki,  55,  61 

Bloch,  52,  116 

Blum,  133,  154 

Boldyreff,  82 

Borchardt,  5,  154 

Bornstein,  161 

Bredig,  31,  125 

Briicke,  14,  25,  28 

Buchner,  E.,  5,  11,30,41, 

Buchner,  H.,  5,  11,  30,  41 

Bunge,  39,  132,  135 


Cannon,  jj 
96,  99,       Chittenden,  106 
Chodat,  125,  132 
Cohnheim,  J.,  4,  28,  60 
Cohnheim,  O.,  8,  49,  58,  84,  85, 
86,  89,  90,  91,  92,  95,  104,  109, 
116,  136,  141,  146,  148,  156, 

^158 

Connstein,  71,  106,  120 

Cremer,  41,  155 

Dakin,  15,  47,  113,  115,  132,  133 
Deetjen,  in,  163 
Dreyfus,  86 
During,  136 

Ebstein,  67 
Edsall,  97 
Ehrlich,  F.,  155 
Ehrlich,  P.,  18,  133 
Ellenberger,  77 
Ellinger,  157 
Embden,  149,  154 
Emmerling,  40 
Ewald,  92 

Fisher,  E,.  40,  44,  46,  119 

Fletcher,  160 

Frank,  136 
71,145       Friedenthal,  27,  117 
,  145  Friedmann,  133 

v.  Furth,  129 
167 


168 


INDEX  OF  AUTHORS 


Geelmuyden,  154 
Geppert,  161 
Geret,  105 
Gignon,  60 
Grafe,  34 
Griitzner,  67,  77 
Gumlich,  99 

Hahn,  5,  57,  105,  145,  151 
Hamburger,  65,  76,  95,  119 
Hammarsten,  87,  162 
Hedin,  8,  57,  58,  113,  115 
Heidenhain,  68 
Hekma,  65,  95 
Henderson,  158 
Herting,  103 
Herzen,  86 

Croft-Hill,  40 
Hochbauer,  101 
Hopkins,  160 
Hoppe-Seyler,  132 
Hoyer,  71 

Ibrahim,  78,  79 
Iwanoff,  20 

Jaeggy,  97 

Jacoby,  8,  14,  28,  87,  in,  115, 

125,  126,  127,  131 
Jaquet,  126 
Jochmann,  55,  113 
Jones,  122 

Kastle,  41,  132 

Kehrer,  140 

Knoop,  51 

Kossel,  5,  15,  19,  39,  47,  107,  113, 

115,  118 
Kiihne,  13,  68,  85,  92,  119 
Kurajeff,  90 


Kutscher,  95,  99,  101,  105,  106, 
113.  120 

Langstein,  97 
Laquer,  81 
Lea,  59 
Leathes,  113 
Lehmann,  122 
Leo,  25 

Lewandowski,  117 
Levene,  90 
Lintwarew,  68,  69 
Lockemann,  55 
Loevenhart,  41,  70,  132 
Lohmann,  101 
Lohrisch,  102 
Liidy,  120 
Lusk,  155 
Liithje,  155 

Maase,  133 

Macfadyen,  5 

Magnus,  70,  141 

Magnus-Levy,  154 

Malfatti,  90 

Malloizel,  7 

Masing,  131 

Matthes,  no 

Mays,  55 

Medigreceanu,  117 

Meissenheimer,  150 

Meltzer,  21,  62 

Mendel,  76,  79,  80,  104,  106, 117 

v.  Mering,  76 

Meyer,  E.,  157 

Milroy,  99 

Miquel,  121 

Mitchell,  79 

Moore,  82 

Morawitz,  147,  162 


INDEX    OF    AUTHORS 


169 


Morawski,  132 
Morgenroth,  63 
Miiller,  113      , 
Musculus,  76 

Nakayama,  99 
Nencki,  42,  69,  120 
Neubauer,  134,  144 
Neumeister,  106,  117 
Niebel,  119 
Nierenstein,  104 
Niirnberg,  90 

Okunew,  90 
Opie,  9,  in 

Palladin,  151 

Powlow,  1,  56,  68,  69,  78,  87,  95 

Pekelharing,  18 

Pfeffer,  53 

Pinkussohn,  117 

Pletnew,  109,  116,  136 

Plimmer,  78 

Popoff,  99 

Pottevin,  35,  41 

Pringsheim,  6 

Quincke,  ioi 

Raudnitz,  125 
Riesser,  47 
Rockwood,  82,  117 
Rona,  52 
Roselle,  14 
Rowland,  5,  8,  115 
Rubner,  136 

Sachs,  63,  99 
Salaskin,  85,  90,  95 
Salkowski,  8,  105,  115,  126 
Sauerland,  131 


Sawitsch,  68,  87 

Sawjalow,  90 

Schierbeck,  27 

Schittenhelm,  97,  99,  122 

Schmiedebcrg,  39,  49 

Schmidt,  A.,  92 

Schneider,  129 

Schonbein,  124 

Schulze,  129 

Schumm,  8,  114,  120 

Schulze,  106 

Schumoff-Simanowski,  101,  120 

Schiitz,  102 

Seeman,  95 

Sieber,  101,  120 

Shiga,  122 

Slowtzoff,  127 

van  Slyke,    90 

Spitzer,  125 

Starling,  1 

Steudel,  128 

Stoklasa,  151 

O'Sullivan,  56 

Taiki,  80 

Takamura,  90,  113 
Tappeiner,  102 
Teruuchi,  49,  96 
Thierf  elder,  155 
Thompson,  56 
Tichomirow,  21 
Tobler,  8,  59,  85,  89 

v.  Undransky,  157 
Umber,  99 
Underhill,  76,  117 

Vernon,  10,  88,  115,  136,  147, 

152 
Vines,  107 


170 

Volhard,  81 


INDEX  OF  AUTHORS 


Wakeman,  III 

Walther,  78 

Warburg,  132,  146,  149 

Wartenberg,  71 

Weinland,  63,  79,  80,  no,  135 


Weiss,  118 
Wiechowski,  7,  122 
Wittich,  2 

Zemplen,  6 
v.  Zeynek,  101 
Zuntz,  69,  136,  161 


GENERAL    INDEX 


ACETO ACETIC  ACID,  1 33 

Acetoacetic  acid  formation,  154 

Acetone  formation,  149,  154 

Acetone  yeast,  7 

/3-acids,  51 

Action  antiseptics  upon  enzymes 

29 
Activators,  67 
Adsorption,  12,  13 
Albumose,  85 
Aldehydase,  125 
Alkaptonuria,  143 
Amidophenol,  128 
Amyl  alcohols,  formation,  155 
Antiferments,  63 
Antipepsin,  64 
Arginase,  15,  47,  97 
Atoxyl,  133 
Autolysis,  9 

Bacterial  enzymes,  105 
Bile  salts,  69 
Blood  platlets,  163 
Bromelin,  106 

Carbohydrate-splitting  en- 
zymes, 74 

Catalase,  124 

Catalyzers,  32 

Chemical  properties  of  enzymes, 
27 

Cellulose,  digestion  of,  102 

Configuration  of  sugars,  45 


Cysteine,  conversion  into  taur- 
ine, 157 

Dawerhefe,  7 

Energy  development,  139 
Endoenzymes,  1 
Endotrypsin,  105,  150 
Enzymes  as  Catalyzers,  31 
Enzymes  of  cancer,  117 
Enzymes  of  invertebrates,  103 
Enzymes  of  plants,  106 
Enterokinase,  68 
Erepsin,  96 

Ethyl  butyrate  synthesis,  41 
Extracellular  enzymes,  1 

Ferments,  iii 
Fibrin  ferments,  162 
Furfurogallin,  127 

Gastric  erepsin,  90 

Gastric  lipase,  81 

Glycerin  transformation,  155 

Glycolytic  enzyme  of  muscles,  7 

Glycyl-glycine,  48 

Glycuronic  acid  formation,  155 

Guaiaconic  acid,  130 

Heat  of  combustion,  139 
Hippuric  acid,  48 
Homogentisic  acid,  143,  158 
Hormon  of  the  pancreas,  70 


171 


172 


GENERAL    INDEX 


Hydrolytic  enzymes  of  the  ali- 
mentary canal,  73 

Hydrolytic  enzymes  of  blood 
and  tissues,  119 

Hydrolytic  enzymes  of  tissues, 
108 

Individual  enzymes,  73 
Influence  of    dissociation  prod- 
ucts, 58 
Ink-bag  of  Sepia,  129 
Intestinal  lipase,  82 
Intracellular  enzymes,  1 
Inverting  enzymes,  78 
Isolactose,  41 
Isomaltose,  40 

Laccase,  71,  127 

Lactacidase,  150 

Lactic  acid  formation,  158 

Law  of  Schultz,  23 

Lecithase,  100 

Leucoprotease,  112 

Lienoprotease,  113 

Lime    salts,    relation    to    blood 

coagulation,  153 
Lipases  of  the  alimentary  canal, 

81 
Lipase  of  the  pancreas  and  liver, 

69 
Lipase  of  ricinus  seeds,  71 
Lymphoprotease,   112 
Lysine,   conversion  to  cadaver- 

ine,  157 

Magen  strasse,  91 
Manganese,  as  activator,  71,  131 
Metabolism  enzymes,  145 
Methods  of  obtaining  enzymes,  1 
Methyl  glucosides,  45 


Mode  of  action,  55 

Nomenclature,  iii 
Nuclease,  100,  121 
Nucleins,  99 

Optical  activity,  44 
Ornithine,  conversion  to  putres- 

cine,  157 
Oxidases,  124 
Oxidations,  138 
/3-oxybutyric  acid,  133 
/5-oxybutyric     acid     formation, 

154 
Oxyurushic  acid,  127 

Pancreatic  lipase,  82 

Papain,  106 

Peptids,  49,  60 

Phenylalanine,  conversion  into 
homogentisic  acid,  157 

Phosphates,  relation  to  zymase, 
27,  71 

Plastein,  90 

Properties  of  enzymes,  20 

Protrolytic  enzymes  of  the  ali- 
mentary canal,  84 

Proteolytic  enzymes  of  the  blood, 
9,  no 

Proteolytic  enzymes  of   tissues, 

115 
Purification  of  enzymes,  1 1 

Quinone,  127 

Reaction  conditions,  24 
Reasons  why  enzymes  are  not 

catalyzers,  33 
Rennin,  87 

Reversible  reactions,  36 
Reversible  action  of  enzymes,  39 


GENERAL    INDEX 


173 


Respiratory  quotient,  136 

Secretin,  77 

Specificity  of  enzymes,  66 

Steapsins,  35 

Sugar    formation    from    amino 

acids,   155 
Synthesis  of  fats,  41 

Temperature,  23,  55 
Thrombin,  162 
Thrombokinase,  163 
Time  velocity,  37 
Tragersubstanz,  18 


Trypsinogen,  68 
Tryptic  digestion,  92 
Tyrosinase,  128 

Tyrosine,  conversion  into  homo- 
gentisic  acid,  157 

Urase,  121 

Uricolytic  enzymes,  122 

Urushic  acid,  127 

Xyloside,  47 

Zymase,  7,  145 
Zymogens,  67 


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BRIDGES    AND    ROOFS,    HYDRAULICS,    MATERIALS    OP    ENGINEER- 
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6 


BRIDGES  AND  ROOFS. 

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Greene — Arches  in  Wood,  Iron,  and  Stone 8vo,     2  50 

Bridge  Trusses 8vo,     2  50 

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Grimm — Secondary  Stresses  in  Bridge  Trusses 8vo,     2  50 

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Symmetrical  Masonry  Arches 8vo,     2  50 

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13 


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MATERIALS  OF  ENGINEERING. 


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14 


*2 

50 

*2 

50 

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00 

3 

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50 

*2 

50 

7 

50 

1 

00 

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*1 

25 

*1 

50 

*1 

50 

*1 

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4 

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*1 

00 

2 

00 

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$3 

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Tables  of   Minerals,   Including  the  Use  of   Minerals  and  Statistics  of 

Domestic  Production 8vo,     1  00 

Pirsson — Rocks  and  Rock  Minerals 12mo,  *2  50 

Richards — Synopsis  of  Mineral  Characters 12mo,  mor.,  *1_25 

18 


Ries—  Building  Stones  and  Clay  Products 8vo,*S3  00 

Clays:  Their  Occurrence,  Properties  and  Uses 8vo,  *5  00 

and  Leighton — History  of  the  Clay-working  Industry    of    the  United 

States » 8vo,  *2  50 

Rowe — Practical  Mineralogy  Simplified 12mo,  *1   25 

TlLLMAN — Text-book  of  Important  Minerals  and  Rocks 8vo,  *2*  00 

Washington — Manual  of  the  Chemical  Analysis  of  Rocks 8vo,     2  00 


MINING. 

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Brunswig — Explosives.      (Munroe  and  Kibler.) Ready  Fall,  1912 

CRANE— Gold  and  Silver 8vo,  *5  00 

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8vo,  mor.,  *5  00 

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8vo,  mor.,  *4  00 

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8vo,  *4  00 

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Ihlseng — Manual  of  Mining 8vo,     5  00 

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Wilson — Hydraulic  and  Placer  Mining 12mo,     2  50 

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Association  of  State  and   National  Food  and   Dairy  Departments, 

Hartford  Meeting,  1906 8vo,     3  00 

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19 


SI 

50 

*1 

50 

2 

50 

1 

00 

1 

00 

1 

00 

1 

00 

*0 

50 

2 

00 

*1 

50 

4 

00 

2 

50 

5 

00 

2 

00 

3 

00 

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50 

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00 

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00 

Prescott   and   Winslow — Elements   of   Water  Bacteriology,   with   Special 

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Price — Handbook  on  Sanitation 12mo, 

Richards — Conservation  by  Sanitation 8vo, 

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